Pretargeting methods and compounds

ABSTRACT

Methods, compounds, compositions and kits that relate to pretargeted delivery of diagnostic and therapeutic agents are disclosed. In particular, methods for radiometal labeling of biotin and for improved radiohalogenation of biotin, as well as related compounds, are described. Also, clearing agents, anti-ligand-targeting moiety conjugates, target cell retention enhancing moieties and additional methods are discussed.

TECHNICAL FIELD

[0001] The present invention relates to methods, compounds, compositionsand kits useful for delivering to a target site a targeting moiety thatis conjugated to one member of a ligand/anti-ligand pair. Afterlocalization and clearance of the targeting moiety conjugate, direct orindirect binding of a diagnostic or therapeutic agent conjugate at thetarget site occurs. Methods for radiometal labeling of biotin and forimproved radiohalogenation of biotin, as well as the related compounds,are also disclosed. Also, clearing agents, anti-ligand-targeting moietyconjugates, target cell retention enhancing moieties and additionalmethods are set forth.

BACKGROUND OF THE INVENTION

[0002] Conventional cancer therapy is plagued by two problems. Thegenerally attainable targeting ratio (ratio of administered doselocalizing to tumor versus administered dose circulating in blood orratio of administered dose localizing to tumor versus administered dosemigrating to bone marrow) is low. Also, the absolute dose of radiationor therapeutic agent delivered to the tumor is insufficient in manycases to elicit a significant tumor response. Improvement in targetingratio or absolute dose to tumor is sought.

SUMMARY OF THE INVENTION

[0003] The present invention is directed to diagnostic and therapeuticpretargeting methods, moieties useful therein and methods of makingthose moieties. Such pretargeting methods are characterized by animproved targeting ratio or increased absolute dose to the target cellsites in comparison to conventional cancer therapy.

[0004] The present invention describes chelate-biotin compounds andradiohalogenated biotin compounds useful in diagnostic-and therapeuticpretargeting methods. The present invention also provides targetingmoiety-ligand, such as biotin, compounds useful in diagnostic andtherapeutic pretargeting methods. Selection of moieties andmethodologies used to enhance internalization (of chemotherapeuticdrugs, for example) or to enhance retention at the target cell surface(of radionuclides, for example) is also discussed.

[0005] In addition, the present invention provides targetingmoiety-anti-ligand, such as avidin or streptavidin, compounds useful indiagnostic and therapeutic pretargeting methods. Otherligand-anti-ligand systems including the zinc finger protein-dsDNAfragment binding pair are also contemplated. Preparation andpurification of such anti-ligand-targeting moiety compounds are alsodiscussed.

[0006] The present invention also provides clearing agents to facilitatethe removal of circulating targeting moiety-ligand (two-step) ortargeting moiety-anti-ligand (two-step) or anti-ligand (three-step) fromthe mammalian recipient. Preferred clearing agents are classifiable asgalactose-based and non-galactose-based. Within each category,preferable clearing agents are polymeric or protein based. Particulateagents, extracorporeal procedures and in vivo devices are alsocontemplated for use in the practice of the present invention.

[0007] Also, the present invention is directed to methods usingstreptavidin as an anti-ligand to enhance retention of radionuclide attarget cell sites, with pretargeting protocols constituting one suchmethod. More specifically, these embodiments of the present inventioninvolve either (1) targeting moiety-streptavidin-radionuclide (with theradionuclide bound to streptavidin directly or through a chelate orlinker), as well as (2) targeting moiety-biotin administered prior tostreptavidin-radionuclide, or (3) biotin-radionuclide bound to apretargeted streptavidin containing molecule.

[0008] The present invention further provides pretargeting methodsemploying intraarterial or other local administration of the therapeuticmoiety-containing molecule to achieve greater localization thereof toartery-supplied target cell populations. Other methods of the presentinvention involve administration of short duration bone marrowprotecting agents or vascular permeability enhancing agents prior toradionuclide-ligand molecule or radionuclide-anti-ligand moleculeadministration. Further, monovalent targeting moieties, such as Fv orFab antibody fragments, are useful in the inventive pretargetingmethods. Delivery of other, non-radioactive therapeutic agents, such aschemotherapeutic drugs, anti-tumor agents (e.g., cytokines) and toxins,to target cells using the pretargeting methods of the present inventionis also described.

BRIEF DESCRIPTION OF THE DRAWINGS

[0009]FIG. 1 illustrates blood clearance of biotinylated antibodyfollowing intravenous administration of avidin.

[0010]FIG. 2 depicts radiorhenium tumor uptake in a three-steppretargeting protocol, as compared to administration of radiolabeledantibody (conventional means involving antibody that is covalentlylinked to chelated radiorhenium).

[0011]FIG. 3 depicts the tumor uptake profile of NR-LU-10-streptavidinconjugate (LU-10-StrAv) in comparison to a control profile of nativeNR-LU-10 whole antibody.

[0012]FIG. 4 depicts the tumor uptake and blood clearance profiles ofNR-LU-10-streptavidin conjugate.

[0013]FIG. 5 depicts the rapid clearance from the blood ofasialoorosomucoid in comparison with orosomucoid in terms of percentinjected dose of I-125-labeled protein.

[0014]FIG. 6 depicts the 5 minute limited biodistribution ofasialoorosomucoid in comparison with orosomucoid in terms of percentinjected dose of I-125-labeled protein.

[0015]FIG. 7 depicts NR-LU-10-streptavidin conjugate blood clearanceupon administration of three controls (◯, , ▪) and two doses of aclearing agent (

, □) at 25 hours post-conjugate administration.

[0016]FIG. 8 shows limited biodistribution data for LU-10-StrAvconjugate upon administration of three controls (Groups 1, 2 and 5) andtwo doses of clearing agent (Groups 3 and 4) at two hours post-clearingagent administration.

[0017]FIG. 9 depicts NR-LU-10-streptavidin conjugate serum biotinbinding capability at 2 hours post-clearing agent administration.

[0018]FIG. 10 depicts NR-LU-10-streptavidin conjugate blood clearanceover time upon administration of a control (◯) and three doses of aclearing agent (∇, Δ, □) at 24 hours post-conjugate administration.

[0019]FIG. 11A depicts the blood clearance of LU-10-StrAv conjugate uponadministration of a control (PBS) and three doses (50, 20 and 10 μg) ofclearing agent at two hours post-clearing agent administration.

[0020]FIG. 11B depicts LU-10-StrAv conjugate serum biotin bindingcapability upon administration of a control (PBS) and three doses (50,20 and 10 μg) of clearing agent at two hours post-clearing agentadministration.

[0021]FIG. 12 depicts the prolonged tumor retention ofNR-LU-10-streptavidin conjugate (▴) relative to NR-LU-10 whole antibody(Δ) over time.

[0022]FIG. 13 depicts the prolonged liver retention of a pre-formedcomplex of NR-LU-10-biotin (◯; chloramine T labeled with I-125)complexed with streptavidin (; PIP-I-131 labeled).

[0023]FIG. 14 depicts the prolonged liver retention of Biotin-PIP-I-131label relative to the streptavidin-NR-LU-10-(PIP-I-125) label.

[0024]FIG. 15A depicts tumor uptake for increasing doses of PIP-Biocytinin terms of % ID/G.

[0025]FIG. 15B depicts tumor uptake for increasing doses of PIP-Biocytinover time in terms of pMOL/G.

[0026]FIG. 16A depicts tumor versus blood localization of a 0.5 μg doseof PIP-Biocytin over time in terms of % ID/G.

[0027]FIG. 16B depicts tumor versus blood localization of a 0.5 μg doseof PIP-Biocytin in terms of % ID.

[0028]FIG. 17A depicts tumor uptake of LU-10-StrAv and PIP-Biocytin overtime in terms of % ID/G.

[0029]FIG. 17B depicts blood clearance of LU-10-StrAv and PIP-Biocytinover time in terms of % ID/G.

[0030]FIG. 18 depicts PIP-Biocytin:LU-10-StrAv ratio in tumor and bloodover time.

DETAILED DESCRIPTION OF THE INVENTION

[0031] Prior to setting forth the invention, it may be helpful to setforth definitions of certain terms to be used within the disclosure.

[0032] Targeting moiety: A molecule that binds to a defined populationof cells. The targeting moiety may bind a receptor, an oligonucleotide,an enzymatic substrate, an antigenic determinant, or other binding sitepresent on or in the target cell population. Antibody is used throughoutthe specification as a prototypical example of a targeting moiety. Tumoris used as a prototypical example of a target in describing the presentinvention.

[0033] Ligand/anti-ligand pair: A complementary/anti-complementary setof molecules that demonstrate specific binding, generally of relativelyhigh affinity. Exemplary ligand/anti-ligand pairs include zinc fingerprotein/dsDNA fragment, enzyme/inhibitor, hapten/antibody,lectin/carbohydrate, ligand/receptor, and biotin/avidin. Biotin/avidinis used throughout the specification as a prototypical example of aligand/anti-ligand pair.

[0034] Anti-lipand: As defined herein, an “anti-ligand” demonstrateshigh affinity, and preferably, multivalent binding of the complementaryligand. Preferably, the anti-ligand is large enough to avoid rapid renalclearance, and contains sufficient multivalency to accomplishcrosslinking and aggregation of targeting moiety-ligand conjugates.Univalent anti-ligands are also contemplated by the present invention.Anti-ligands of the present invention may exhibit or be derivitized toexhibit structural features that direct the uptake thereof, e.g.,galactose residues that direct liver uptake. Avidin and streptavidin areused herein as prototypical anti-ligands.

[0035] Avidin: As defined herein, “avidin” includes avidin, streptavidinand derivatives and analogs thereof that are capable of high affinity,multivalent or univalent binding of biotin.

[0036] Ligand: As defined herein, a “ligand” is a relatively small,soluble molecule that exhibits rapid serum, blood and/or whole bodyclearance when administered intravenously in an animal or human. Biotinis used as the prototypical ligand.

[0037] Active Agent: A diagnostic or therapeutic agent (“the payload”),including radionuclides, drugs, anti-tumor agents, toxins and the like.Radionuclide therapeutic agents are used as prototypical active agents.

[0038] N_(x)S_(t) Chelates: As defined herein, the term “N_(x)S_(y)chelates” includes bifunctional chelators that are capable of (i)coordinately binding a metal or radiometal and (ii) covalently attachingto a targeting moiety, ligand or anti-ligand. Particularly preferredN_(x)S_(y) chelates have. N₂S₂ and N₃S cores. Exemplary N_(x)S_(y)chelates are described in Fritzberg et al., Proc. Natl. Acad. Sci. USA85:4024-29, 1988; in Weber et al., Bioconj. Chem. 1:431-37, 1990; and inthe references cited therein, for instance.

[0039] Pretargeting: As defined herein, pretargeting involves targetsite localization of a targeting moiety that is conjugated with onemember of a ligand/anti-ligand pair; after a time period sufficient foroptimal target-to-non-target accumulation of this targeting moietyconjugate, active agent conjugated to the opposite member of theligand/anti-ligand pair is administered and is bound (directly orindirectly) to the targeting moiety conjugate at the target site(two-step pretargeting). Three-step and other related methods describedherein are also encompassed.

[0040] Clearing Agent: An agent capable of binding, complexing orotherwise associating with an administered moiety (e.g., targetingmoiety-ligand, targeting moiety-anti-ligand or anti-ligand alone)present in the recipient's circulation, thereby facilitating circulatingmoiety clearance from the recipient's body, removal from bloodcirculation, or inactivation thereof in circulation. The clearing agentis preferably characterized by physical properties, such as size,charge, configuration or a combination thereof, that limit clearingagent access to the population of target cells recognized by a targetingmoiety used in the same treatment protocol as the clearing agent.

[0041] Target Cell Retention: The amount of time that a radionuclide orother therapeutic agent remains at the target cell surface or within thetarget cell. Catabolism of conjugates or molecules containing suchtherapeutic agents appears to be primarily responsible for the loss oftarget cell retention.

[0042] Conjugate: A conjugate encompasses chemical conjugates(covalently or non-covalently bound), fusion proteins and the like.

[0043] Permeability Enhancing Moiety: An agent capable of increasing thepermeability at a target site characterized by a three dimensionalcellular matrix. Exemplary permeability enhancing moieties function byone or more of the following mechanisms: inducing gaps in theendothelium of venules through action on the postcapillary bed; inducingsuch gaps through action on the entire capillary bed; disruptingcell-to-cell associations; mediating target cell inflammatory responses;or the like.

[0044] Intercellular Junction: An area of interacting adjacent plasmamembranes. Intercellular junctions can be categorized functionally into:(1) adhering junctions that hold cells tightly together (for example,desmosomes); (2) impermeable junctions that hold cells tightly togetherand prevent leakage of molecules between cells (i.e., tight junctions);and (3) communicating junctions that mediate passage of small moleculesbetween adjacent cells (for instance, gap junctions).

[0045] Immunogen: A substance which is capable, under appropriateconditions, of inducing a specific immune response and of reacting withthe products of that response (e.g., a specific antibody, specificallysensitized T-lymphocytes or both).

[0046] Hapten Immunogen: A specific protein-free substance which has achemical configuration such that it can interact with specific combininggroups on an antibody or with the recognition site on a T-lymphocyte butwhich, unlike antigenic determinants, does not itself elicit an immuneresponse (e.g., a detectable T-cell response or the formation of adetectable amount of antibody). When coupled with a carrier protein, itdoes elicit an immune response.

[0047] Lymphokine. Soluble protein mediators released by certainlymphocytes, which in turn can regulate other cell-mediated immunefunctions, such as lymphocyte transformation, macrophage activation orcytotoxicity on other cells.

[0048] Mitogen. A substance that induces mitosis and celltransformation, especially lymphocyte transformation.

[0049] A recognized disadvantage associated with in vivo administrationof targeting moiety-radioisotopic conjugates for imaging or therapy islocalization of the attached radioactive agent at both non-target andtarget sites. Until the administered radiolabeled conjugate clears fromthe circulation, normal organs and tissues are transitorily exposed tothe attached radioactive agent. For instance, radiolabeled wholeantibodies that are administered in vivo exhibit relatively slow bloodclearance; maximum target site localization generally occurs 1-3 dayspost-administration. Generally, the longer the clearance time of theconjugate from the circulation, the greater the radioexposure ofnon-target organs.

[0050] These characteristics are particularly problematic with humanradioimmunotherapy. In human clinical trials, the long circulatinghalf-life of radioisotope bound to whole antibody causes relativelylarge doses of radiation to be delivered to the whole body. Inparticular, the bone marrow, which is very radiosensitive, is thedose-limiting organ of non-specific toxicity.

[0051] In order to decrease radioisotope exposure of non-target tissue,potential targeting moieties generally have been screened to identifythose that display minimal non-target reactivity, while retaining targetspecificity and reactivity. By reducing non-target exposure (and adversenon-target localization and/or toxicity), increased doses of aradiotherapeutic conjugate may be administered; moreover, decreasednon-target accumulation of a radiodiagnostic conjugate leads to improvedcontrast between background and target.

[0052] Therapeutic drugs, administered alone or as targeted conjugates,are accompanied by similar disadvantages. Again, the goal isadministration of the highest possible concentration of drug (tomaximize exposure of target tissue), while remaining below the thresholdof unacceptable normal organ toxicity (due to non-target tissueexposure). Unlike radioisotopes, however, therapeutic drugs need to betaken into a target cell to exert a cytotoxic effect. In the case oftargeting moiety-therapeutic drug conjugates, it would be advantageousto combine the relative target specificity of a targeting moiety with ameans for enhanced target cell internalization of the targetingmoiety-drug conjugate.

[0053] In contrast, enhanced target cell internalization isdisadvantageous if one administers diagnostic agent-targeting moietyconjugates. Internalization of diagnostic conjugates results in cellularcatabolism and degradation of the conjugate. Upon degradation, smalladducts of the diagnostic agent or the diagnostic agent per se may bereleased from the cell, thus eliminating the ability to detect theconjugate in a target-specific manner.

[0054] One method for reducing non-target tissue exposure to adiagnostic or therapeutic agent involves “pretargeting” the targetingmoiety at a target site, and then subsequently administering a rapidlyclearing diagnostic or therapeutic agent conjugate that is capable ofbinding to the “pretargeted” targeting moiety at the target site. Adescription of some embodiments of the pretargeting technique may befound in U.S. Pat. No. 4,863,713 (Goodwin et al.).

[0055] A typical pretargeting approach (“three-step”) is schematicallydepicted below.

[0056] Briefly, this three-step pretargeting protocol featuresadministration of an antibody-ligand conjugate, which is allowed tolocalize at a target site and to dilute in the circulation. Subsequentlyadministered anti-ligand binds to the antibody-ligand conjugate andclears unbound antibody-ligand conjugate from the blood. Preferredanti-ligands are large and contain sufficient multivalency to accomplishcrosslinking and aggregation of circulating antibody-ligand conjugates.The clearing by anti-ligand is probably attributable to anti-ligandcrosslinking and/or aggregation of antibody-ligand conjugates that arecirculating in the blood, which leads to complex/aggregate clearance bythe recipient's RES (reticuloendothelial system). Anti-ligand clearanceof this type is preferably accomplished with a multivalent molecule;however, a univalent molecule of sufficient size to be cleared by theRES on its own could also be employed. Alternatively, receptor-basedclearance mechanisms, e.g., Ashwell receptor hexose, e.g., galactose,mannose or the like, residue recognition mechanisms, may be responsiblefor anti-ligand clearance. Such clearance mechanisms are less dependentupon the valency of the anti-ligand with respect to the ligand than theRES complex/aggregate clearance mechanisms. It is preferred that theligand-anti-ligand pair displays relatively high affinity binding.

[0057] A diagnostic or therapeutic agent-ligand conjugate that exhibitsrapid whole body clearance is then administered. When the circulationbrings the active agent-ligand conjugate in proximity to the targetcell-bound antibody-ligand-anti-ligand complex, anti-ligand binds thecirculating active agent-ligand conjugate and produces anantibody-ligand: anti-ligand: ligand-active agent “sandwich” at thetarget site. Because the diagnostic or therapeutic agent is attached toa rapidly clearing ligand (rather than antibody, antibody fragment orother slowly clearing targeting moiety), this technique promisesdecreased non-target exposure to the active agent.

[0058] Alternate pretargeting methods eliminate the step of parenterallyadministering an anti-ligand clearing agent. These “two-step” proceduresfeature targeting moiety-ligand or targeting moiety-anti-ligandadministration, followed by administration of active agent conjugated tothe opposite member of the ligand-anti-ligand pair. As an optional step“1.5” in the two-step pretargeting methods of the present invention, aclearing agent (preferably other than ligand or anti-ligand alone) isadministered to facilitate the clearance of circulating targetingmoiety-containing conjugate.

[0059] In the two-step pretargeting approach, the clearing agentpreferably does not become bound to the target cell population, eitherdirectly or through the previously administered and target cell boundtargeting moiety-anti-ligand or targeting moiety-ligand conjugate. Anexample of two-step pretargeting involves the use of biotinylated humantransferrin as a clearing agent for avidin-targeting moiety conjugate,wherein the size of -the clearing agent results in liver clearance oftransferrin-biotin-circulating avidin-targeting moiety complexes andsubstantially precludes association with the avidin-targeting moietyconjugates bound at target cell sites. (See, Goodwin, D. A., Antibod.Immunoconj. Radiopharm., 4: 427-34, 1991).

[0060] The two-step pretargeting approach overcomes certaindisadvantages associated with the use of a clearing agent in athree-step pretargeted protocol. More specifically, data obtained inanimal models demonstrate that in vivo anti-ligand binding to apretargeted targeting moiety-ligand conjugate (i.e., the cell-boundconjugate) removes the targeting moiety-ligand conjugate from the targetcell. One explanation for the observed phenomenon is that themultivalent anti-ligand crosslinks targeting moiety-ligand conjugates onthe cell surface, thereby initiating or facilitating internalization ofthe resultant complex. The apparent loss of targeting moiety-ligand fromthe cell might result from internal degradation of the conjugate and/orrelease of active agent from the conjugate (either at the cell surfaceor intracellularly). An alternative explanation for the observedphenomenon is that permeability changes in the target cell's membraneallow increased passive diffusion of any molecule into the target cell.Also, some loss of targeting moiety-ligand may result from alteration inthe affinity by subsequent binding of another moiety to the targetingmoiety-ligand, e.g., anti-idiotype monoclonal antibody binding causesremoval of tumor bound monoclonal antibody.

[0061] The present invention recognizes that this phenomenon (apparentloss of the targeting moiety-ligand from the target cell) may be used toadvantage with regard to in vivo delivery of therapeutic agentsgenerally, or to drug delivery in particular. For instance, a targetingmoiety may be covalently linked to both ligand and therapeutic agent andadministered to a recipient. Subsequent administration of anti-ligandcrosslinks targeting moiety-ligand-therapeutic agent tripartiteconjugates bound at the surface, inducing internalization of thetripartite conjugate (and thus the active agent). Alternatively,targeting moiety-ligand may be delivered to the target cell surface,followed by administration of anti-ligand-therapeutic agent.

[0062] In one aspect of the present invention, a targetingmoiety-anti-ligand conjugate is administered in vivo; upon targetlocalization of the targeting moiety-anti-ligand conjugate (i.e., andclearance of this conjugate from the circulation), an activeagent-ligand conjugate is parenterally administered. This methodenhances retention of the targeting moiety-anti-ligand: ligand-activeagent complex at the target cell (as compared with targetingmoiety-ligand: anti-ligand: ligand-active agent complexes and targetingmoiety-ligand: anti-ligand-active agent complexes). Although avariety-of ligand/anti-ligand pairs may be suitable for use within theclaimed invention, a preferred ligand/anti-ligand pair is biotin/avidin.

[0063] In a second aspect of the invention, radioiodinated biotin andrelated methods are disclosed. Previously, radioiodinated biotinderivatives were of high molecular weight and were difficult tocharacterize. The radioiodinated biotin described herein is a lowmolecular weight compound that has been easily and well characterized.

[0064] In a third aspect of the invention, a targeting moiety-ligandconjugate is administered in vivo; upon target localization of thetargeting moiety-ligand conjugate (i.e., and clearance of this conjugatefrom the circulation), a drug-anti-ligand conjugate is parenterallyadministered. This two-step method hot only provides pretargeting of thetargeting moiety conjugate, but also induces internalization of thesubsequent targeting moiety-ligand-anti-ligand-drug complex within thetarget cell. Alternatively, another embodiment provides a three-stepprotocol that produces a targeting moiety-ligand: anti-ligandligand-drug complex at the surface, wherein the ligand-drug conjugate isadministered simultaneously or within a short period of time afteradministration of anti-ligand (i.e., before the targetingmoiety-ligand-anti-ligand complex has been removed from the target cellsurface). Additional internalization methodologies are contemplated bythe present invention and are discussed herein.

[0065] In a fourth aspect of the invention, methods for radiolabelingbiotin with technetium-99m, rhenium-186 and rhenium-188 are disclosed.Previously, biotin derivatives were radiolabeled with indium-111 for usein pretargeted immunoscintigraphy (for instance, Virzi et al., Nucl.Med. Biol. 18:719-26, 1991; Kalofonos et al., J. Nucl. Med. 31: 1791-96,1990; Paganelli et al., Canc. Res. 51:5960-66, 1991). However, ^(99m)Tcis a particularly preferred radionuclide for immunoscintigraphy due to(i) low cost, (ii) convenient supply and (iii) favorable nuclearproperties. Rhenium-186 displays chelating chemistry very similar to^(99m)Tc, and is considered to be an excellent therapeutic radionuclide(i.e., a 3.7 day half-life and 1.07 MeV maximum particle that is similarto ¹³¹I). Therefore, the claimed methods for technetium and rheniumradiolabeling of biotin provide numerous advantages.

[0066] The present invention is also directed to radiolabeling withyttrium-90, lutetium-177, sumarium-153, and other appropriate +3 metals.Y-90 is a particularly preferred radionuclide for therapy, because itexhibits favorable nuclear properties including high specific activity,long path length with respect to deposition of radiation in tissue, highequilibrium dose constant and favorable half-life properties. Morespecifically, the beta emission of Y-90 (Beta_(av)=0.937 MeV) is one ofthe most energetic of all beta emitters. The X₉₀ value of Y-90 is 5.34mm (i.e., 90% of the energy emitted grom a point source is absorbed in asphere of 5.34 mm radius). Y-90 has a high equilibrium dose constant ormean energy/nuclear transition, Delta=1.99 Rad-gram/microcurie-hour, anda 64 hour half-life suitable for targeted therapy. Y-90 can bemanufactured at high specific activity and is available as a generatorproduct. Specific advantages of Y-90 are (1) that it has the capabilityto kill neighboring target cells not directly targeted by thepretargeted targeting moiety-ligand or targeting moiety-anti-ligandconjugate and (2) that more radiation is deposited per microcurielocalized than for other beta emitters of lower mean particle energy(provided that a sufficiently large target volume is available).

[0067] Lu-177 is a particularly preferred radionuclide for targetednuclide therapy, since it has a moderately energetic beta emission(Beta_(av)=0.140 MeV); it is available in high specific activity; itsradiochemical production is efficient.; it emits two gammas of idealenergy and abundance for imaging (208 keV, 11% and 113 keV, 7%); and ithas a relatively long half-life (161 hours). The X₉₀ for Lu-177 is 0.31mm, i.e., 90% of the energy emitted form a point source is absorbed in asphere of radius 0.31 mm. Lu-177 has an equilibrium dose constant ormean energy/nuclear transition of 0.31 Rad-gram/microcuries-hour and anadequate half-life to serve as a targeted therapeutic radionuclide.Specific advantages of Lu-177 are (1) that its emitted energy isefficiently absorbed in smaller targeted tumor volumes such asmetastatic tumor foci or involved lymph nodes and (2) that its longphysical half-life makes optimal use of the tumor retention property ofthe pretargeting delivery method.

[0068] The “targeting moiety” of the present invention binds to adefined target cell population, such as tumor cells. Preferredtargeting~moieties useful in this regard include antibody and antibodyfragments, peptides, and hormones. Proteins corresponding to known cellsurface receptors (including low density lipoproteins, transferrin andinsulin), fibrinolytic enzymes, anti-HER2, platelet binding proteinssuch as annexins, and biological response modifiers (includinginterleukin, interferon, erythropoietin and colony-stimulating factor)are also preferred targeting moieties. Also, anti-EGF receptorantibodies, which internalize following binding to the receptor andtraffic to the nucleus to an extent, are preferred targeting moietiesfor use in the present invention to facilitate delivery of Augeremitters and nucleus binding drugs to target cell nuclei.

[0069] Oligonucleotides, e.g., antisense oligonucleotides that arecomplementary to portions of target cell nucleic acids (DNA or RNA), arealso useful as targeting moieties in the practice of the presentinvention. Oligonucleotides binding to cell surfaces are also useful.Analogs of the above-listed targeting moieties that retain the capacityto bind to a defined target cell population may also be used within theclaimed invention. In addition, synthetic targeting moieties may bedesigned.

[0070] Functional equivalents of the aforementioned molecules are alsouseful as targeting moieties of the present invention. One targetingmoiety functional equivalent is a “mimetic” compound, an organicchemical construct designed to mimic the proper configuration and/ororientation for targeting moiety-target cell binding. Another targetingmoiety functional equivalent is a short polypeptide designated as a“minimal” polypeptide, constructed using computer-assisted molecularmodeling and mutants having altered binding affinity, which minimalpolypeptides exhibit the binding affinity of the targeting moiety.

[0071] Preferred targeting moieties of the present invention areantibodies (polyclonal or monoclonal), peptides, oligonucleotides or thelike. Polyclonal antibodies useful in the practice of the presentinvention are polyclonal (Vial and Callahan, Univ. Mich. Med. Bull., 20:284-6, 1956), affinity-purified polyclonal or fragments thereof (Chao etal., Res. Comm. in Chem. Path. & Pharm., 9: 749-61, 1974).

[0072] Monoclonal antibodies useful in the practice of the presentinvention include whole antibody and fragments thereof. Such monoclonalantibodies and fragments are producible in accordance with conventionaltechniques, such as hybridoma synthesis, recombinant DNA techniques andprotein synthesis. Useful monoclonal antibodies and fragments may bederived from any species (including humans) or may be formed as chimericproteins which employ sequences from more than one species. See,generally, Kohler and Milstein, Nature, 256: 495-97, 1975; Eur. J.Immunol., 6: 511-19, 1976.

[0073] Human monoclonal antibodies or “humanized” murine antibody arealso useful as targeting moieties in accordance with the presentinvention. For example, murine monoclonal antibody may be “humanized” bygenetically recombining the nucleotide sequence encoding the murine Fvregion (i.e., containing the antigen binding sites) or thecomplementarity determining regions thereof with the nucleotide sequenceencoding a human constant domain region and an Fc region, e.g., in amanner similar to that disclosed in European Patent Application No.0,411,893 A2. Some murine residues may also be retained within the humanvariable region framework domains to ensure proper target site bindingcharacteristics. Humanized targeting moieties are recognized to decreasethe immunoreactivity of the antibody or polypeptide in the hostrecipient, permitting an increase in the half-life and a reduction inthe possibility of adverse immune reactions.

[0074] Another preferred targeting moiety of the present invention areannexins and other platelet binding proteins, such as PAP-1 (PlacentalAnticoagulant Protein or Annexin V). Annexins are (with the exception ofannexin II) single chain, non-glycosylated proteins of approximately 36kilodaltons. Annexins possess a number of biological properties based onthe principle of calcium ion binding. Investigations have shown thatannexins bind with high affinity to membrane lipids in the presence ofmicromolar quantities of calcium. In the presence of calcium, theseproteins have an especially high affinity for negatively chargedphospholipids such as phosphatidylserine or phosphatidylinosine.

[0075] Annexins exert anti-coagulatory effects. In a manner analogous toanti-inflammatory mechanisms, coagulation inhibition is mediated by thebinding of annexins to negatively charged surface phospholipids (i.e.,to platelets) which binding blocks the activation of clotting factors bysuch negatively charged surface phospholipids. Annexins localize totarget sites rapidly, i.e., in a matter of minutes, but remainscirculating in the serum for a longer time period.

[0076] As a result of these properties, annexins may be employed intwo-step or three-step pretargeting protocols for the diagnosis of bloodclots or the treatment of blood clots associated with indications, suchas DVT (deep vein thrombosis), PE (pulmonary embolism), heart attack,stroke and the like. Exemplary diagnostic and treatment protocolsemploying the two-step pretargeting approach are set forth below tofurther elucidate this aspect of the present invention.

[0077] For the visualization of blood clots associated with a number ofpathological conditions, a chemical conjugate or a fusion protein ofannexin with avidin or streptavidin is administered to a recipient forwhom such a diagnosis is desired. The annexin portion of the conjugatelocalizes rapidly to target sites characterized by negatively chargedsurface phospholipids, such as blood clots. As a consequence of therapid target site uptake, a clearing agent, such as those discussedherein, may be administered after the passage of a short time (rangingfrom about 5 minutes to about 1 hour, with within about 15 minutespreferred). The clearing agent serves to rapidly diminish the serumlevel of the annexin-anti-ligand conjugate. Next, biotin labeled with animaging radionuclide, such as Tc-99m for example, is administered.Biotin directs the localization of the administered moiety to thepreviously localized annexin-anti-ligand. Non-target bound radiolabeledbiotin is rapidly cleared from the recipient. Consequently, imaging ofthe target sites proceeds with minimal exposure of non-target sites toradioactivity. This approach offers the advantages of speed,facilitating imaging of difficult target sites such as lung clots, andimproved clot to blood ratios.

[0078] For therapeutic applications involving blood clots associatedwith a number of pathological conditions, a chemical conjugate or afusion protein of annexin with avidin or streptavidin is administered toa recipient for whom such treatment is desired. A clearing agent, suchas those discussed herein, may be administered after the passage of ashort time (ranging from about 5 minutes to about 1 hour, with withinabout 15 minutes preferred) to diminish the serum level of theannexin-anti-ligand conjugate. Biotin conjugated with therapeuticagents, such as fibrolytic agents, tissue plasminogen activator,thrombolytic agents (e.g., streptokinase, anisoylated plasminogenstreptokinase activator complex) and the like, are deliverable in thismanner. Such a dosing regimen may be repeated, for example, once dailyfor a number of weeks to address the recipient's physiologicalcondition. Consequently, delivery of a therapeutic agent to target sitesproceeds with minimal exposure of non-target sites to that agent.

[0079] An example of an annexin useful in the practice of the presentinvention is Annexin V which was isolated by Bohn in 1979 from humanplacenta, a rich source of annexins, and termed Placenta Protein 4(PP4). Annexin V consists of four domains, with each made up of fivealpha helices wherein the alpha helices serve as connecting elementsbetween the domains. From the side, the molecule appears crown-like withfive calcium binding sites on its convex surface, through whichannexin-phospholipid interactions are mediated. Other annexins havingthe characteristics described above are also useful in the practice ofthe present invention. Annexin V has been expressed in E. coli.

[0080] Types of active agents (diagnostic or therapeutic) useful hereininclude toxins, anti-tumor agents, drugs and radionuclides. Several ofthe potent toxins useful within the present invention consist of an Aand a B chain. The A chain is the cytotoxic portion and the B chain isthe receptor-binding portion of the intact toxin molecule (holotoxin).Because toxin B chain may mediate non-target cell binding, it is oftenadvantageous to conjugate only the toxin A chain to a targeting moiety.However, while elimination of the toxin B chain decreases non-specificcytotoxicity, it also generally leads to decreased potency of the toxinA chain-targeting protein conjugate, as compared to the correspondingholotoxin-targeting moiety conjugate.

[0081] Preferred toxins in this regard include holotoxins, such asabrin, ricin, modeccin, Pseudomonas exotoxin A, Diphtheria toxin,pertussis toxin and Shiga toxin; and A chain or “A chain-like”molecules, such as ricin A chain, abrin A chain, modeccin A chain, theenzymatic portion of Pseudomonas exotoxin A, Diphtheria toxin A chain,the enzymatic portion of pertussis toxin, the enzymatic portion of Shigatoxin, gelonin, pokeweed antiviral protein, saporin, tritin, barleytoxin and snake venom peptides. Ribosomal inactivating proteins (RIPs),naturally occurring protein synthesis inhibitors that lack translocatingand cell-binding ability, are also suitable for use herein.

[0082] Preferred drugs suitable for use herein include conventionalchemotherapeutics, such as vinblastine, doxorubicin, bleomycin,methotrexate, 5-fluorouracil, 6-thioguanine, cytarabine,cyclophosphamide and cis-platinum, as well as other conventionalchemotherapeutics as described in Cancer: Principles and Practice ofOncology, 2d ed., V. T. DeVita, Jr., S. Hellman, S. A. Rosenberg, J. B.Lippincott Co., Philadelphia, Pa., 1985, Chapter 14. A particularlypreferred drug within the present invention is a trichothecene.

[0083] Trichothecenes are drugs produced by soil fungi of the classFungi imperfecti or isolated from Baccharus megapotamica (Bamburg, J. R.Proc. Molec. Subcell. Biol. 8:41-110, 1983; Jarvis & Mazzola, Acc. Chem.Res. 15:338-395, 1982). They appear to be the most toxic molecules thatcontain only carbon, hydrogen and oxygen (Tamm, C. Fortschr. Chem. Org.Naturst. 31:61-117, 1974). They are all reported to act at the level ofthe ribosome as inhibitors of protein synthesis at the initiation,elongation, or termination phases.

[0084] There are two broad classes of trichothecenes: those that haveonly a central sesquiterpenoid structure and those that have anadditional macrocyclic ring (simple and macrocyclic trichothecenes,respectively). The simple trichothecenes may be subdivided into threegroups (i.e., Group A, B, and C) as described in U.S. Pat. Nos.4,744,981 and 4,906,452 (incorporated herein by reference).Representative examples of Group A simple trichothecenes include:Scirpene, Roridin C, dihydrotrichothecene, Scirpen-4,8-diol, Verrucarol,Scirpentriol, T-2 tetraol, pentahydroxyscirpene, 4-deacetylneosolaniol,trichodermin, deacetylcalonectrin, calonectrin, diacetylverrucarol,4-monoacetoxyscirpenol, 4,15-diacetoxyscirpenol,7-hydroxydiacetoxyscirpenol, 8-hydroxydiacetoxy-scirpenol (Neosolaniol),7,8-dihydroxydiacetoxyscirpenol, 7-hydroxy-8-acetyldiacetoxyscirpenol,8-acetylneosolaniol, NT-1, NT-2, HT-2, T-2, and acetyl T-2 toxin.Representative examples of Group B simple trichothecenes include:Trichothecolone, Trichothecin, deoxynivalenol, 3-acetyldeoxynivalenol,5-acetyldeoxynivalenol, 3,15-diacetyldeoxynivalenol, Nivalenol,4-acetylnivalenol (Fusarenon-X), 4,15-idacetylnivalenol,4,7,15-triacetylnivalenol, and tetra-acetylnivalenol. Representativeexamples of Group C simple trichothecenes include: Crotocol andCrotocin. Representative macrocyclic trichothecenes include VerrucarinA, Verrucarin B, Verrucarin J (Satratoxin C), Roridin A, Roridin D,Roridin E (Satratoxin D), Roridin H, Satratoxin F, Satratoxin G,Satratoxin H, Vertisporin, Mytoxin A, Mytoxin C, Mytoxin B, Myrotoxin A,Myrotoxin B, Myrotoxin C, Myrotoxin D, Roritoxin A, Roritoxin B, andRoritoxin D. In addition, the general “trichothecene” sesquiterpenoidring structure is also present in compounds termed “baccharins” isolatedfrom the higher plant Baccharis megapotamica, and these are described inthe literature, for instance as disclosed by Jarvis et al. (Chemistry ofAlleopathy, ACS Symposium Series No. 268: ed. A. C. Thompson, 1984, pp.149-159).

[0085] Experimental drugs, such as mercaptopurine, N-methylformamide,2-amino-1,3,4-thiadiazole, melphalan, hexamethylmelamine, galliumnitrate, 3% thymidine, dichloromethotrexate, mitoguazone, suramin,bromodeoxyuridine, iododeoxyuridine, semustine,1-(2-chloroethyl)-3-(2,6-dioxo-3-piperidyl)-1-nitrosourea,N,N′-hexamethylene-bis-acetamide, azacitidine, dibromodulcitol, Erwiniaasparaginase, ifosfamide, 2-mercaptoethane sulfonate, teniposide, taxol,3-deazauridine, soluble Baker's antifol, homoharringtonine,cyclocytidine, acivicin, ICRF-187, spiromustine, levamisole,chlorozotocin, aziridinyl benzoquinone, spirogermanium, aclarubicin,pentostatin, PALA, carboplatin, amsacrine, caracemide, iproplatin,misonidazole, dihydro-5-azacytidine, 4′-deoxy-doxorubicin, menogaril,triciribine phosphate, fazarabine, tiazofurin, teroxirone, ethiofos,N-(2-hydroxyethyl)-2-nitro-1H-imidazole-1-acetamide, mitoxantrone,acodazole, amonafide, fludarabine phosphate, pibenzimol, didemnin B,merbarone, dihydrolenperone, flavone-8-acetic-acid, oxantrazole,ipomeanol, trimetrexate, deoxyspergualin, echinomycin, anddideoxycytidine (see NCI Investigational Drugs, Pharmaceutical Data1987, NIH Publication No. 88-2141, Revised November 1987) are alsopreferred.

[0086] Radionuclides useful within the present invention includegamma-emitters, positron-emitters, Auger electron-emitters, X-rayemitters and fluorescence-emitters, with beta- or alpha-emitterspreferred for therapeutic use. Radionuclides are well-known in the artand include ¹²³I, ¹²⁵I, ¹³⁰I, ¹³¹I, ¹³³i, ¹³⁵I, ⁴⁷Sc, ⁷²As, ⁷²Se, ⁹⁰Y,⁸⁸Y, ⁹⁷Ru, ¹⁰⁰Pd, ^(101m)Rh, ¹¹⁹Sb, ¹²⁸Ba, ¹⁹⁷Hg, ²¹¹At, ²¹²Bi, ¹⁵³Sm,¹⁶⁹Eu, ²¹²Pb, ¹⁰⁹Pd, ¹¹¹In, ⁶⁷Ga, ⁶⁸Ga, ⁶⁷Cu, ⁷⁵Br, ⁷⁶Br, ⁷⁷Br,^(99m)Tc, ¹¹C, ¹³N, ¹⁵O and ¹⁸F. Preferred therapeutic radionuclidesinclude ¹⁸⁸Re, ¹⁸⁶Re, ²⁰³Pb, ²¹²Pb, ²¹²Bi, ¹⁰⁹Pd, ⁶⁴Cu, ⁶⁷Cu, ⁹⁰Y, ¹²⁵I,¹³¹I, ⁷⁷Br, ²¹¹At, ⁹⁷Ru, ¹⁰⁵Rh, ¹⁹⁸Au and ¹⁹⁹Ag or ¹⁷⁷Lu.

[0087] Other anti-tumor agents are administrable in accordance with thepresent invention. Exemplary anti-tumor agents include cytokines, suchas IL-2, tumor necrosis factor or the like, lectin inflammatory responsepromoters (selecting), such as L-selectin, E-selectin, P-selectin or thelike, and like molecules.

[0088] Ligands suitable for use within the present invention includebiotin, haptens, lectins, epitopes, dsDNA fragments, enzyme inhibitorsand analogs and derivatives thereof. Useful complementary anti-ligandsinclude avidin (for biotin), carbohydrates (for lectins), antibody,fragments or analogs thereof, including mimetics (for haptens andepitopes) and zinc finger proteins (for dsDNA fragments) and enzymes(for enzyme inhibitors). Preferred ligands and anti-ligands bind to eachother with an affinity of at least about k_(D)≧10⁻⁹ M.

[0089] As mentioned above, zinc finger protein/dsDNA is aligand-anti-ligand binding pair contemplated by the present invention.Zinc finger proteins are a class of proteins containing repeatingsubunits of approximately 30 amino acids that bind to specific promoterregions on dsDNA and aid in transcription. These proteins are alsocapable of binding to dsRNA with unknown function. Like antibodies, zincfinger proteins are a family of proteins consisting of variable andconserved regions. Zinc finger proteins, as a class, contain from about2 to about 39 repeating subunits (fingers), with each such fingercapable of binding specific nucleotide sequences. Each finger is capableof binding to a “cleft” in the conformation of the dsDNA/RNA structurethat is dependent upon nucleotide sequence. The present inventorshypothesize that greater numbers of subunits correspond to greateraffinity and specificity. In addition, zinc is incorporated into theproteins within each subunit and is important in maintaining properconformation for dsDNA binding.

[0090] To illustrate the use of the zinc finger protein/dsDNA bindingpair in pretargeting protocols, a two-step approach is described below.This ligand-anti-ligand binding pair may also be used in three-steppretargeting protocols of the present invention. For two-steppretargeting, a zinc finger protein or a dsDNA fragment is conjugated toa targeting moiety in any convenient manner therefor, including, forexample, conjugation methods described herein.

[0091] Several methods have been developed to conjugate DNA oroligonucleotides to proteins. One simple method is oxidation of thesugar moiety of the DNA. The resultant aldehyde groups are available forreaction with amines on the protein. The amines generated by suchreactions are then reduced by reaction with a reducing agent such asNaCNBH₄ to form the more stable amine bonds.

[0092] Another protein-oligonucleotide conjugation method involvesinitial oxidation of the sugar residue on DNA using an oxidizing agentsuch as NaIO₄. The dialdehyde formed is treated with 5-pyridylcystaminehydrochloride and NaCNBH₃. This dithiopyridine derivative can then bereacted with tributylphosphine to generate the thiol adduct. Reaction ofthe free thiol-bearing DNA adduct with a maleimide-derivatized proteinresults in a thioether oligonucleotide-protein conjugate. See, forexample, Kuijpers et al., “Specific Recognition ofAntibody-Oligonucleotide Conjugates by Radiolabeled AntisenseNucleotides: A Novel Approach for Two-Step Radioimmunotherapy ofCancer,” Bioconiugate Chem., A: 94-102 (1993).

[0093] The conjugates are administered to a recipient by a route chosenin view of the patient's physical condition and known or anticipatedillness. Following target site localization of the administeredconjugate (or clearing agent treatment as described herein),administration of the other member of the ligand pair conjugated to atherapeutic agent (e.g., radionuclide, drug or anti-tumor agent) isundertaken. The administered therapeutic agent-containing conjugatebinds to the previously localized targeting agent containing conjugate.Therapeutic agent-containing conjugate that does not bind at the targetsite will be eliminated from the recipient's body at a much more rapidrate than a monoclonal antibody-therapeutic agent conjugate would beremoved. Moreover, such therapeutic agent-containing conjugates can bechemically altered for more rapid excretion, if necessary, to reducenon-target exposure to the therapeutic agent.

[0094] A schematic of the administered components and ultimate in vivoformed “sandwich” for a monoclonal antibody-zinc fingerprotein/dsDNA-therapeutic agent two-step pretargeting protocol are shownbelow.

[0095] Use of the zinc finger protein/dsDNA fragment ligand/anti-ligandbinding pair offers advantages with respect to immunogenicity. Thetargeting agent used in a pretargeting protocol may be human orhumanized, and all of the other administered components (exclusive ofthe therapeutic agent) are human in origin. An additional advantage ofthis approach is that zinc finger proteins can be engineered toaccommodate specific, high affinity interactions with synthetic orcloned dsDNA fragments, thereby eliminating non-specific interactions ofthe administered components with normal tissue.

[0096] One embodiment of this aspect of the present invention involvesthe use of pretargeting approaches to target double stranded DNA itself.Such protocols of the present invention are useful, for example, forgene therapy, delivery of tumor suppressive DNA, deleted genes and thelike. More specifically, a targeting moiety-zinc finger protein ispretargeted to a target site. Subsequently, a dsDNA fragment that iscomplementary to the zinc finger protein is injected and allowed to bindto the localized zinc finger protein. Because zinc finger protein-DNAinteraction is a normal cell physiologic mechanism, DNA will bedelivered free of encumbering chemistry to the cellular milieu. ThedsDNA is then internalized by the target cell via the normal mechanismstherefor, i.e., via endocytosis or the like.

[0097] One component to be administered in a preferred two-steppretargeting protocol involving the biotin-avidin ligand-anti-ligandsystem is a targeting moiety-avidin or a targeting moiety-streptavidinconjugate. The preferred targeting moiety useful in these embodiments ofthe present invention is a monoclonal antibody. Protein-proteinconjugations are generally problematic due to the formation ofundesirable byproducts, including high molecular weight and cross-linkedspecies, however. A non-covalent synthesis technique involving reactionof biotinylated antibody with streptavidin has been reported to resultin substantial byproduct formation. Also, at least one of the fourbiotin binding sites on the streptavidin is used to link the antibodyand streptavidin, while another such binding site may be stericallyunavailable for biotin binding due to the configuration of thestreptavidin-antibody conjugate.

[0098] Thus, covalent streptavidin-antibody conjugation is preferred,but high molecular weight byproducts are often obtained. The degree ofcrosslinking and aggregate formation is dependent upon several factors,including the level of protein derivitization using heterobifunctionalcrosslinking reagents. Sheldon et al., Appl. Radiat. Isot. 43:1399-1402, 1992, discuss preparation of covalent thioether conjugates byreacting succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate(SMCC)-derivitized antibody and iminothiolane-derivitized streptavidin.

[0099] Streptavidin-proteinaceous targeting moiety conjugates arepreferably prepared as described in Example XI below, with thepreparation involving the steps of: preparation of SMCC-derivitizedstreptavidin; preparation of DTT-reduced proteinaceous targeting moiety;conjugation of the two prepared moieties; and purification of themonosubstituted conjugate. The purified fraction is preferably furthercharacterized by one or more of the following techniques: HPLC sizeexclusion, SDS-PAGE, immunoreactivity, biotin binding capacity and invivo studies.

[0100] Alternatively, thioether conjugates useful in the practice of thepresent invention may be formed using other thiolating agents, such asSPDP, iminothiolate, SATA or the like, or other thio-reactiveheterobifunctional cross linkers, such asm-maleimidobenzoyl-N-hydroxysuccinimide ester,N-succinimidyl(4-iodoacetyl)aminobenzoate or the like.

[0101] Streptavidin-proteinaceous targeting moiety conjugates of thepresent invention can also be formed by conjugation of a lysine epsilonamino group of one protein with a maleimide-derivitized form of theother protein. For example, at pH 8-10, lysine epsilon amino moietiesreact with protein maleimides, prepared, for instance, by treatment ofthe protein with SMCC, to generate stable amine covalent conjugates. Inaddition, conjugates can be prepared by reaction of lysine epsilon aminomoieties of one protein with aldehyde functionalities of the otherprotein. The resultant imine bond is reducible to generate thecorresponding stable amine bond. Aldehyde functionalities may begenerated, for example, by oxidation of protein sugar residues or byreaction with aldehyde-containing heterobifunctional cross linkers.

[0102] Another method of forming streptavidin-targeting moietyconjugates involves immobilized iminobiotin that binds SMCC-derivitizedstreptavidin. In this conjugation/purification method, the reversiblebinding character of iminobiotin (immobilized) to streptavidin isexploited to readily separate conjugate from the unreacted targetingmoiety. Iminobiotin binding can be reversed under conditions of lower pHand elevated ionic strength, e.g., NH₂OAc, pH 4 (50 mM) with 0.5 M NaCl.

[0103] For streptavidin, for example, the conjugation/purificationproceeds as follows:

[0104] SMCC-derivitized streptavidin is bound to immobilized iminobiotin(Pierce Chemical Co., St. Louis, Mo.), preferably in column format;

[0105] a molar excess (with respect to streptavidin) of DTT-reducedantibody (preferably free of reductant) is added to the nitrogen-purged,phosphate-buffered iminobiotin column wherein the SMCC-streptavidin isbound (DTT-reduced antibody will saturate the bound SMCC-streptavidin,and unbound reduced antibody passing through the column can be reused);

[0106] the column is washed free of excess antibody; and

[0107] a buffer that lowers the pH and increases ionic strength is addedto the column to elute streptavidin-antibody conjugate in pure form.

[0108] As indicated above, targeting moiety-mediated ligand-anti-ligandpretargeting involves the localization of either targeting moiety-ligandor targeting moiety-anti-ligand at target tissue. Often, peak uptake tosuch target tissue is achieved before the circulating level of targetingmoiety-containing conjugate in the blood is sufficiently low to permitthe attainment of an optimal target-to-non-target conjugate ratio. Toobviate this problem, two approaches are useful. The first approachallows the targeting moiety-containing conjugate to clear from the bloodby “natural” or endogenous clearance mechanisms. This method iscomplicated by variations in systemic clearance of proteins and byendogenous ligand or anti-ligand. For example, endogenous biotin mayinterfere with the preservation of biotin binding sites on astreptavidin-targeting moiety conjugate.

[0109] The second approach for improving targeting moiety-ligand ortargeting moiety-anti-ligand conjugate target-to-blood ratio “chases”the conjugate from the circulation through in vivo complexation ofconjugate with a molecule constituting or containing the complementaryanti-ligand or ligand. When biotinylated antibodies are used as aligand-targeting moiety conjugate, for example, avidin forms relativelylarge aggregated species upon complexation with the circulatingbiotinylated antibody, which aggregated species are rapidly cleared fromthe blood by the RES uptake. See, for example, U.S. Pat. No. 4,863,713.One problem with this method, however, is the potential forcross-linking and internalizing tumor-bound biotinylated antibody byavidin.

[0110] When avidin-targeting moiety conjugates are employed,poly-biotinylated transferrin has been used to form relatively largeaggregated species that are cleared by RES uptake. See, for example,Goodwin, J. Nucl. Med. 33(10):1816-18, 1992). Poly-biotinylatedtransferrin also has the potential for cross-linking and internalizingtumor-bound avidinylated-targeting moiety, however. In addition, both“chase” methodologies involve the prolonged presence of aggregatedmoieties of intermediate, rather than large, size (which are not clearedas quickly as large size particles by RES uptake), thereby resulting inserum retention of subsequently administered ligand-active agent oranti-ligand-active agent. Such serum retention unfavorably impactsthe-target cell-to-blood targeting ratio.

[0111] The present invention provides clearing agents of protein andnon-protein composition having physical properties facilitating use forin vivo complexation and blood clearance of anti-ligand/ligand (e.g.,avidin/biotin)-targeting moiety (e.g., antibody) conjugates. Theseclearing agents are useful in improving the target:blood ratio oftargeting moiety conjugate. Other applications of these clearing agentsinclude lesional imaging or therapy involving blood clots and the like,employing antibody-active agent delivery modalities. For example,efficacious anti-clotting agent provides rapid target localization andhigh target:non-target targeting ratio. Active agents administered inpretargeting protocols of the present invention using efficient clearingagents are targeted in the desirable manner and are, therefore, usefulin the imaging/therapy of conditions such as pulmonary embolism and deepvein thrombosis.

[0112] Clearing agents useful in the practice of the present inventionpreferably exhibit one or more of the following characteristics:

[0113] rapid, efficient complexation with targeting moiety-ligand (oranti-ligand) conjugate in vivo;

[0114] rapid clearance from the blood of targeting moiety conjugatecapable of binding a subsequently administered complementary anti-ligandor ligand containing molecule;

[0115] high capacity for clearing (or inactivating) large amounts oftargeting moiety conjugate; and

[0116] low immunogenicity.

[0117] Preferred clearing agents include hexose-based and non-hexosebased moieties. Hexose-based clearing agents are molecules that havebeen derivatized to incorporate one or more hexoses (six carbon sugarmoieties) recognized by Ashwell receptors. Exemplary of such hexoses aregalactose, maonose and the like. Galactose is the prototypical clearingagent hexose derivative for the purposes of this description. Thus,galactose-based and non-galactose based molecules are discussed below.

[0118] Protein-type galactose-based clearing agents include proteinshaving endogenous exposed galactose residues or which have beenderivitized to expose or incorporate such galactose residues. Exposedgalactose residues direct the clearing agent to rapid clearance byendocytosis into the liver through specific receptors. therefor (Ashwellreceptors). These receptors bind the clearing agent, and induceendocytosis into the hepatocyte, leading to fusion with a lysosome andrecycle of the receptor back to the cell surface. This clearancemechanism is characterized by high efficiency, high capacity and rapidkinetics.

[0119] An exemplary clearing agent of theprotein-based/galactose-bearing variety is the asialoorosomucoidderivative of human alpha-1 acid glycoprotein (orosomucoid, molecularweight=41,000. Dal, isoelectric point=1.8-2.7). The rapid clearance fromthe blood of asialoorosomucoid has been documented by Galli, et al., J.of Nucl. Med. Allied Sd. 32(2): 110-16, 1988.

[0120] Treatment of orosomucoid with neuraminidase removes sialic acidresidues, thereby exposing galactose residues. Other such derivitizedclearing agents include, for example, galactosylated albumin,galactosylated-IgM, galactosylated-IgG, asialohaptoglobin, asialofetuin,asialoceruloplasmin and the like.

[0121] Human serum albumin (HSA), for example, may be employed in aclearing agent of the present invention as follows:

[0122] (Hexose)_(m)—Human Serum Albumin (HSA)—(Ligand)_(n), wherein n isan integer from 1 to about 10 and m is an integer from 1 to about 25 andwherein the hexose is recognized by Ashwell receptors.

[0123] In a preferred embodiment of the present invention the ligand isbiotin and the hexose is galactose. More preferably, HSA is derivatizedwith from 10-20 galactose residues and 1-5 biotin residues. Still morepreferably, HSA clearing agents of the present invention are derivatizedwith from about 12 to about 15 galactoses and 3 biotins. Derivatizationwith both galactose and biotin are conducted in a manner sufficient toproduce individual clearing agent molecules with a range ofbiotinylation levels that averages a recited whole number, such as 1,biotin. Derivatization with 3 biotins, for example, produces a productmixture made up of individual clearing agent molecules, substantiallyall of which having at least one biotin residue. Derivatization with 1biotin produces a clearing agent product mixture, wherein a significantportion of the individual molecules are not biotin derivatized. Thewhole numbers used in this description refer to the averagebiotinylation of the clearing agents under discussion.

[0124] In addition, clearing agents based upon human proteins,especially human serum proteins such as, for example, orosomucoid andhuman serum albumin, are less immunogenic upon administration into theserum of a human recipient. Another advantage of using asialoorosomucoidis that human orosomucoid is commercially available from, for example,Sigma Chemical Co, St. Louis, Mo.

[0125] One way to prevent clearing agent compromise of target-boundconjugate through direct complexation is through use of a clearing agentof a size sufficient to render the clearing agent less capable ofdiffusion into the extravascular space and binding to target-associatedconjugate. This strategy is useful alone or in combination with theaforementioned recognition that exposed galactose residues direct rapidliver uptake. This size-exclusion strategy enhances the effectiveness ofnon-galactose-based clearing agents of the present invention. Thecombination (exposed galactose and size) strategy improves theeffectiveness of “protein-type” or “polymer-type” galactose-basedclearing agents.

[0126] Galactose-based clearing agents include galactosylated,biotinylated proteins (to remove circulating streptavidin-targetingmoiety conjugates, for example) of intermediate molecular weight(ranging from about 40,000 to about 200,000 Dal), such as biotinylatedasialoorosomucoid, galactosyl-biotinyl-human serum albumin or othergalactosylated and biotinylated derivatives of non-immunogenic solublenatural proteins, as well as biotin- and galactose-derivitizedpolyglutamate, polylysine, polyarginine, polyaspartate and the like.High molecular weight moieties (ranging from about 200,000 to about1,000,000 Dal) characterized by poor target access, includinggalactosyl-biotinyl-IgM or -IgG (approximately 150,000 Dal) molecules,as well as galactose- and biotin-derivitized transferrin conjugates ofhuman serum albumin, IgG and IgM molecules and the like, can also beused as clearing agents of the claimed invention. Chemically modifiedpolymers of intermediate or high molecular weight (ranging from about40,000 to about 1,000,000 Dal), such as galactose- andbiotin-derivitized dextran, hydroxypropylmethacrylamide polymers,polyvinylpyrrolidone-polystyrene copolymers, divinyl ether-maleic acidcopolymers, pyran copolymers, or PEG, also have utility as clearingagents in the practice of the present invention. In addition, rapidlyclearing biotinylated liposomes (high molecular weight moieties withpoor target access) can be derivitized with galactose and biotin toproduce clearing agents for use in the practice of the presentinvention.

[0127] A further class of clearing agents useful in the presentinvention involve small molecules (ranging from about 500 to about10,000 Dal) derivitized with galactose and biotin that are sufficientlypolar to be confined to the vascular space as an in vivo volume ofdistribution. More specifically, these agents exhibit a highly chargedstructure and, as a result, are not readily distributed into theextravascular volume, because they do not readily diffuse across thelipid membranes lining the vasculature. Exemplary of such clearingagents are mono- or poly-biotin-derivitized6,6′-[(3,3′-dimethyl[1,1′-biphenyl]-4,4′-diyl)bis(azo)bis[4-amino-5-hydroxy-1,3-naphthalenedisulfonic acid]tetrasodium salt, mono- orpoly-biotinyl-galactose-derivitized polysulfated dextran-biotin, mono-or poly-biotinyl-galactose-derivitized dextran-biotin and the like.

[0128] The galactose-exposed or -derivitized clearing agents arepreferably capable of (1) rapidly and efficiently complexing with therelevant ligand- or anti-ligand-containing conjugates vialigand-anti-ligand affinity; and (2) clearing such complexes from theblood via the galactose receptor, a liver specific degradation system,as opposed to aggregating into complexes that are taken up by thegeneralized RES system, including the lung and spleen. Additionally, therapid kinetics of galactose-mediated liver uptake, coupled with theaffinity of the ligand-anti-ligand interaction, allow the use ofintermediate or even low molecular weight carriers.

[0129] Non-galactose residue-bearing moieties of low or intermediatemolecular weight (ranging from about 40,000 to about 200,000 Dal)localized in the blood may equilibrate with the extravascular space and,therefore, bind directly to target-associated conjugate, compromisingtarget localization. In addition, aggregation-mediated clearancemechanisms operating through the RES system are accomplished using alarge stoichiometric excess of clearing agent.

[0130] In contrast, the rapid blood clearance of galactose-basedclearing agents used in the present invention prevents equilibration,and the high affinity ligand-anti-ligand binding allows the use of lowstoichiometric amounts of such galactose-based clearing agents. Thisfeature further diminishes the potential for galactose-based clearingagents to compromise target-associated conjugate, because the absoluteamount of such clearing agent administered is decreased.

[0131] Clearing agent evaluation experimentation involving galactose-and biotin-derivatized clearing agents of the present invention isdetailed in Example XXII. Specific clearing agents of the presentinvention that were examined during the Example XXII experimentation are(1) asialoorosomucoid-biotin, (2) human serum albumin derivatized withgalactose and biotin, and (3) a 70,000 dalton molecular weight dextranderivatized with both biotin and galactose. The experimentation showedthat proteins and polymers are derivatizable to contain both galactoseand biotin and that the resultant derivatized molecule is effective inremoving circulating streptavidin-protein conjugate from the serum ofthe recipient. Biotin loading was varied to determine the effects onboth learing the blood pool of circulating avidin-containing conjugateand the ability to deliver a subsequently administered biotinylatedisotope to a target site recognized by the streptavidin-containingconjugate. The effect of relative doses of the administered componentswith respect to clearing agent efficacy was also examined.

[0132] Protein-type and polymer-type non-galactose-based clearing agentsinclude the agents described above, absent galactose exposure orderivitization and the like. These clearing agents act through anaggregation-mediated RES mechanism. In these embodiments of the presentinvention, the clearing agent used will be selected on the basis of thetarget organ to which access of the clearing agent is to be excluded.For example, high molecular weight (ranging from about 200,000 to about1,000,000 Dal) clearing agents will be used when tumor targets or clottargets are involved.

[0133] Another class of clearing agents includes agents that do notremove circulating ligand or anti-ligand/targeting moiety conjugates,but instead “inactivate” the circulating conjugates by blocking therelevant anti-ligand or ligand binding sites thereon. These “cap-type”clearing agents are preferably small (500 to 10,000 Dal) highly chargedmolecules, which exhibit physical characteristics that dictate a volumeof distribution equal to that of the plasma compartment (i.e., do notextravasate into the extravascular fluid volume). Exemplary cap-typeclearing agents are poly-biotin-derivitized6,6′-[(3,3′-dimethyl[1,1′-biphenyl]-4,4′-diyl)bis(azo)bis[4-amino-5-hydroxy-1,3-naphthalene disulfonic acid]tetrasodium salt,poly-biotinyl-derivitized polysulfated dextran-biotin, mono- orpoly-biotinyl-derivitized dextran-biotin and the like.

[0134] Cap-type clearing agents are derivitized with the relevantanti-ligand or ligand, and then administered to a recipient ofpreviously administered ligand/ or anti-ligand/targeting moietyconjugate. Clearing agent-conjugate binding therefore diminishes theability of circulating conjugate to bind any subsequently administeredactive agent-ligand or active agent-anti-ligand conjugate. The ablationof active agent binding capacity of the circulating conjugate increasesthe efficiency of active agent delivery to the target, and increases theratio of target-bound active agent to circulating active agent bypreventing the coupling of long-circulating serum protein kinetics withthe active agent. Also, confinement of the clearing agent to the plasmacompartment prevents compromise of target-associated ligand oranti-ligand.

[0135] Clearing agents of the present invention may be administered insingle or multiple doses. A single dose of biotinylated clearing agent,for example, produces a-rapid decrease in the level of circulatingtargeting moiety-streptavidin, followed by a small increase in thatlevel, presumably caused, at least in part, by re-equilibration oftargeting moiety-streptavidin within the recipient's physiologicalcompartments. A second or additional clearing agent doses may then beemployed to provide supplemental clearance of targetingmoiety-streptavidin. Alternatively, clearing agent may be infusedintravenously for a time period sufficient to clear targetingmoiety-streptavidin in a continuous manner.

[0136] Other types of clearing agents and clearance systems are alsouseful in the practice of the present invention to remove circulatingtargeting moiety-ligand or -anti-ligand conjugate from the recipient'scirculation. Particulate-based clearing agents, for example, arediscussed in Example IX. In addition, extracorporeal clearance systemsare discussed in Example IX. In vivo clearance protocols employingarterially inserted proteinaceous or polymeric multiloop devices arealso described in Example IX.

[0137] One embodiment of the present invention in which rapid actingclearing agents are useful is in the delivery of Auger emitters, such asI-125, I-123, Er-165, Sb-119, Hg-197, Ru-97, Tl-201 and Br-77, ornucleus-binding drugs to target cell nuclei. In these embodiments of thepresent invention, targeting moieties that localize to internalizingreceptors on target cell surfaces are employed to deliver a targetingmoiety-containing conjugate (i.e., a targeting moiety-anti-ligandconjugate in the preferred two-step protocol) to the target cellpopulation. Such internalizing receptors include EGF receptors,transferrin receptors, HER2 receptors, IL-2 receptors, otherinterleukins and cluster differentiation receptors, somatostatinreceptors, other peptide binding receptors and the like.

[0138] After the passage of a time period sufficient to achievelocalization of the conjugate to target cells, but insufficient toinduce internalization of such targeted conjugates by those cellsthrough a receptor-mediated event, a rapidly acting clearing agent isadministered. In a preferred two-step protocol, an activeagent-containing ligand or anti-ligand conjugate, such as a biotin-Augeremitter or a biotin-nucleus acting drug, is administered as soon as theclearing agent has been given an opportunity to complex with circulatingtargeting moiety-containing conjugate, with the time lag betweenclearing agent and active agent administration being less than about 24hours. In this manner, active agent is readily internalized throughtarget cell receptor-mediated internalization. While circulating Augeremitters are thought to be non-toxic, the rapid, specific targetingafforded by the pretargeting protocols of the present inventionincreases the potential of shorter half-life Auger emitters, such asI-123, which is available and capable of stable binding.

[0139] The 1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetra acetic acid(DOTA)-biotin conjugate (DOTA-LC-biotin) depicted below has beenreported to have desirable in vivo biodistribution and is clearedprimarily by renal excretion.

[0140] DOTA may also be conjugated to other ligands or to anti-ligandsin the practice of the present invention.

[0141] Because DOTA strongly binds Y-90 and other radionuclides, it hasbeen proposed for use in radioimmunotherapy. For therapy, it is veryimportant that the radionuclide be stably bound within the DOTA chelateand that the DOTA chelate be stably attached to biotin. Onlyradiolabeled DOTA-biotin conjugates exhibiting those two characteristicsare useful to deliver radionuclides to the targets. Release of theradionuclide from the DOTA chelate or cleavage of the biotin and DOTAconjugate components in serum or at non-target sites renders theconjugate unsuitable for use in therapy.

[0142] Serum stability of DOTA-LC-biotin (where LC refers to the “longchain” linker, including an aminocaproyl spacer between the biotin andthe DOTA conjugate components) shown above, while reported in theliterature to be good, has proven to be problematic. Experimentation hasrevealed that DOTA-LC-biotin is rapidly cleared from the blood andexcreted into the urine as fragments, wherein the biotinamide bondrather than the DOTA-amide bond has been cleaved, as shown below.

[0143] Additional experimentation employing PIP-biocytin conjugatesproduced parallel results as shown below.

[0144] Cleavage of the benzamide was not observed as evidenced by theabsence of detectable quantities of iodobenzoic acid in the serum.

[0145] It appears that the cleavage results from the action of serumbiotinidase. Biotinidase is a hydrolytic enzyme that catalyzes thecleavage of biotin from biotinyl peptides. See, for example,Evangelatos, et al., “Biotinidase Radioassay Using an I-125-BiotinDerivative, Avidin, and Polyethylene Glycol Reagents,” AnalyticalBiochemistry, 196: 385-89, 1991.

[0146] Drug-biotin conjugates which structurally resemble biotinylpeptides are potential substrates for cleavage by plasma biotinidase.Poor in vivo stability therefore limits the use of drug-biotinconjugates in therapeutic applications. The use of peptide surrogates toovercome poor stability of peptide therapeutic agents has been an areaof intense research effort. See, for example, Spatola, Peptide BackboneModification: A Structure-Activity Analysis of Peptide Containing AmideBond Surrogates, “Chemistry and Biochemistry of Amino Acids, Peptidesand Proteins,” vol. 7, Weinstein, ed., Marcel Dekker, New York, 1983;and Kim et al., “A New Peptide Bond Surrogate: 2-Isoxazoline inPseudodipeptide Chemistry,” Tetrahedron Letters, 45: 6811-14, 1991.

[0147] Elimination of the aminocaproyl spacer of DOTA-LC-biotin givesDOTA-SC-biotin (where the SC indicates the “short chain” linker betweenthe DOTA and biotin conjugate components), which molecule is shownbelow:

[0148] DOTA-SC-biotin exhibits significantly improved serum stability incomparison to DOTA-LC-biotin. This result does not appear to beexplainable on the basis of biotinidase activity alone. Theexperimentation leading to this conclusion is summarized in the Tableset forth below. Time Dependent Cleavage of DOTA-Biotin Conjugates %Avidin Binding Y-90-LC Y-90-SC Time at 37° C. PIP-Biocytin DOTA-BiotinDOTA-Biotin  5 Minutes 75% 50% — 15 Minutes 57% 14% — 30 Minutes 31% 12%— 60 Minutes — 0% 98% 20 Hours — 0% 60%

[0149] The difference in serum stability between DOTA-LC-biotin andDOTA-SC-biotin might be explained by the fact that the SC derivativecontains an aromatic amide linkage in contrast to the aliphatic amidelinkage of the LC derivative, with the aliphatic amide linkage beingmore readily recognized by enzymes as a substrate therefor. Thisargument cannot apply to biotinidase, however, because biotinidase veryefficiently cleaves aromatic amides. In fact, it is recognized that thesimplest and most commonly employed biotinidase activity measuringmethod uses N-(d-biotinyl)-4-aminobenzoate (BPABA) as a substrate, withthe hydrolysis of BPABA resulting in the liberation of biotin and4-aminobenzoate (PABA). See, for example, B. Wolf, et al., “Methods inEnzymology,” pp. 103-111, Academic Press Inc., 1990. Consequently, onewould predict that DOTA-SC-biotin, like its LC counterpart, would be abiotinidase substrate. Since DOTA-SC-biotin exhibits serum stability,biotinidase activity alone does not adequately explain why someconjugates are serum stable while others are not. A series ofDOTA-biotin conjugates was therefore synthesized by the presentinventors to determine which structural features conferred serumstability to the conjugates.

[0150] Some general strategies for improving serum stability of peptideswith respect to enzymatic action are the following: incorporation ofD-amino acids, N-methyl amino acids and alpha-substituted amino acids.

[0151] In vivo stable biotin-DOTA conjugates are useful within thepractice of the present invention. In vivo stability imparts thefollowing advantages:

[0152] 1) increased tumor uptake in that more of the radioisotope willbe targeted to the previously localized targeting moiety-streptavidin;and

[0153] 2) increased tumor retention, if biotin is more stably bound tothe radioisotope.

[0154] In addition, the linkage between DOTA and biotin may also have asignificant impact on biodistribution (including normal organ uptake,target uptake and the like) and pharmacokinetics.

[0155] The strategy for design of the DOTA-containing molecules andconjugates of the present invention involved three primaryconsiderations:

[0156] 1) in vivo stability (including biotinidase and general peptidaseactivity resistance), with an initial cut of 100% stability for 1 hour;

[0157] 2) renal excretion; and

[0158] 3) ease of synthesis.

[0159] The DOTA-biotin conjugates of the present invention reflect theimplementation of one or more of the following strategies:

[0160] 1) substitution of the carbon adjacent to the cleavagesusceptible amide nitrogen;

[0161] 2) alkylation of the cleavage susceptible amide nitrogen;

[0162] 3) substitution of the amide carbonyl with an alkyl amino group;

[0163] 4) incorporation of D-amino acids as well as analogs orderivatives thereof; or

[0164] 5) incorporation of thiourea linkages.

[0165] DOTA-biotin conjugates in accordance with the present inventionmay be generally characterized as follows: conjugates that retain thebiotin carboxy group in the structure thereof and those that do not(i.e., the terminal carboxy group of biotin has been reduced orotherwise chemically modified. Structures of such conjugates representedby the following general formula have been devised:

[0166] wherein L may alternatively be substituted in one of thefollowing ways on one of the —CH₂—COOH branches of the DOTA structure:—CH(L)—COOH or —CH₂COOL or —CH₂COL). When these alternative structuresare employed, the portion of the linker bearing the functional group forbinding with the DOTA conjugate component is selected for the capabilityto interact with either the carbon or the carboxy in the branch portionsof the DOTA structure, with the serum stability conferring portion ofthe linker structure being selected as described below.

[0167] In the case where the linkage is formed on the core of the DOTAstructure as shown above, L is selected according to the followingprinciples, with the portion of the linker designed to bind to the DOTAconjugate component selected for the capability to bind to an amine.

[0168] A. One embodiment of the present invention includes linkersincorporating a D-amino acid spacer between a DOTA aniline amine and thebiotin carboxy group shown above. Substituted amino acids are preferredfor these embodiments of the present invention, becausealpha-substitution also confers enzymatic cleavage resistance. ExemplaryL moieties of this embodiment of the present invention may berepresented as follows:

[0169] where R¹ is selected from lower alkyl, lower alkyl substitutedwith hydrophilic groups (preferably, (CH₂)_(n)—OH, (CH₂)_(n)—OSO₃,(CH₂)_(n)—SO₃,

[0170] where n is 1 or 2), glucuronide-substituted amino acids or otherglucuronide derivatives; and

[0171] R² is selected from hydrogen, lower alkyl, substituted loweralkyl (e.g., hydroxy, sulfate, phosphonate or a hydrophilic moiety(preferably OH).

[0172] For the purposes of the present disclosure, the term “loweralkyl” indicates an alkyl group with from one to five carbon atoms.Also, the term “substituted” includes one or several substituent groups,with a single substituent group preferred.

[0173] Preferred L groups of this embodiment of the present inventioninclude the following:

[0174] R¹═CH₃ and R²═H (a D-alanine derivative, with a synthetic schemetherefor shown in Example XXI);

[0175] R¹═CH₃ and R²═CH₃ (an N-methyl-D-alanine derivative);

[0176] R¹═CH₂—OH and R²═H (a D-serine derivative);

[0177] R¹═CH₂OSO₃ and R²═H (a-D-serine-O-sulfate-derivative); and

[0178] and R²═H (a D-serine-O-phosphonate-derivative);

[0179] Other preferred moieties of this embodiment of the presentinvention include molecules wherein R¹ is hydrogen and R²═—(CH₂)_(n)OHor a sulfate or phosphonate derivative thereof and n is 1 or 2 as wellas molecules wherein R¹ is

[0180] Preferred moieties incorporating the glucuronide of D-lysine andthe glucuronide of amino pimelate are shown below as I and II,respectively.

[0181] A particularly preferred linker of this embodiment of the presentinvention is the D-alanine derivative set forth above.

[0182] B. Linkers incorporating alkyl substitution on one or more amidenitrogen atoms are also encompassed by the present invention, with someembodiments of such linkers preparable from L-amino acids. Amide bondshaving a substituted amine moiety are less susceptible to enzymaticcleavage. Such linkers exhibit the following general formula:

[0183] where R⁴ is selected from hydrogen, lower alkyl, lower alkylsubstituted with hydroxy, sulfate, phosphonate or the like and

[0184] R₃ is selected from hydrogen; an amine; lower alkyl; an amino- ora hydroxy-, sulfate- or phosphonate-substituted lower alkyl; aglucuronide or a glucuronide-derivatized amino groups; and

[0185] n ranges from 0-4.

[0186] Preferred linkers of this embodiment of the present inventioninclude:

[0187] R³═H and R⁴═CH₃ when n=4, synthesizable as discussed in ExampleXXI;

[0188] R³═H and R⁴═CH₃ when n=0, synthesizable from N-methyl-glycine(having a trivial name of sarcosine) as described in Example XXI;

[0189] R³═NH₂ and R⁴═CH₃, when n=0;

[0190] R³═H and

[0191] when n=4 (Bis-DOTA-LC-biotin), synthesizable from bromohexanoicacid as discussed in Example XXI; and

[0192] R³═H and

[0193] when n=0 (bis-DOTA-SC-biotin), synthemizable from iminodiaceticacid.

[0194] The synthesis of a conjugate including a linker wherein R³ is Hand R⁴ is —CH₂CH₂OH and n is 0 is also described in Example XXI.Schematically, the synthesis of a conjugate of this embodiment of thepresent invention wherein n is 0, R³ is H and R⁴ is —CH₂—COOH is shownbelow.

[0195] Bis-DOTA-LC-biotin, for example, offers the following advantages:

[0196] 1) incorporation of two DOTA molecules on one biotin moietyincreases the overall hydrophilicity of the biotin conjugate and therebydirects in vivo distribution to urinary excretion; and

[0197] 2) substitution of the amide nitrogen adjacent to the biotincarboxyl group blocks peptide and/or biotinidase cleavage at that site.

[0198] Bis-DOTA-LC-biotin, the glycine-based linker and the N-methylatedlinker where R³═H, R⁴═CH₃, n=4 are particularly preferred linkers ofthis embodiment of the present invention.

[0199] C. Another linker embodiment incorporates a thiourea moietytherein. Exemplary thiourea adducts of the present invention exhibit thefollowing general formula:

[0200] where R⁵ is selected from hydrogen or lower alkyl;

[0201] R⁶ is selected from H and a hydrophilic moiety; and

[0202] n ranges from 0-4.

[0203] Preferred linkers of this embodiment of the present invention areas follows:

[0204] R⁵═H and R⁶═H when n=5;

[0205] R⁵═H and R⁶═COOH when n 32 5; and

[0206] R⁵═CH₃ and R⁶═COOH when n=5.

[0207] The second preferred linker recited above can be prepared usingeither L-lysine or D-lysine.

[0208] Similarly, the third preferred linker can be prepared usingeither N-methyl-D-lysine or N-methyl-L-lysine.

[0209] Another thiourea adduct of minimized lipophilicity is

[0210] which may be formed via the addition of biotinhydrazide(commercially available from Sigma Chemical Co., St. Louis, Mo.) andDOTA-benzyl-isothiocyanate (a known compound synthesized in one stepfrom DOTA-aniline), with the thiourea-containing compound formed asshown below.

[0211] D. Amino acid-derived linkers of the present invention withsubstitution of the carbon adjacent to the cleavage susceptible amidehave the general formula set forth below:

[0212] wherein Z is —(CH₂)₂—, conveniently synthesized form glutamicacid; or

[0213] Z=—CH₂—S—CH₂—, synthesizable from cysteine and iodo-acetic acid;or

[0214] Z=—CH₂—, conveniently synthesized form aspartic acid; or

[0215] Z=—(CH₂)_(n)—C—O—CH₂—, where n ranges from 1-4 and which issynthesizable from serine.

[0216] E. Another exemplary linker embodiment of the present inventionhas the general formula set forth below:

[0217] and n ranges from 1-5.

[0218] F. Another embodiment involves disulfide-containing linkers,which provide a metabolically cleavable moiety (—S—S—) to reducenon-target retention of the biotin-DOTA conjugate. Exemplary linkers ofthis type exhibit the following formula:

[0219] where n and n, preferably range between 0 and 5.

[0220] The advantage of using conditionally cleavable linkers is animprovement in target/non-target localization of the active agent.Conditionally cleavable linkers include enzymatically cleavable linkers,linkers that are cleaved under acidic conditions, linkers that arecleaved under basic conditions and the like. More specifically, use oflinkers that are cleaved by enzymes, which are present in non-targettissues but reduced in amount or absent in target tissue, can increasetarget cell retention of active agent relative to non-target cellretention. Such conditionally cleavable linkers are useful, for example,in delivering therapeutic radionuclides to target cells, because suchactive agents do not require internalization for efficacy, provided thatthe linker is stable at the target cell surface or protected from targetcell degradation.

[0221] G. Ether, thioether, ester and thioester linkers are also usefulin the practice of the present invention, because such linkages are acidcleavable and therefore facilitate improved non-target retention.Exemplary linkers of this type have the following general formula:

[0222] where X is O or S; and

[0223] Q is a bond, a methylene group, a —CO— group or—CO—(CH₂)_(n)—NH—; and

[0224] n ranges from 1-5.

[0225] Other such linkers have the general formula: —CH₂—X-Q, where Qand X are defined as set forth above.

[0226] H. Another amino-containing linker of the present invention isstructured as follows:

[0227] preferably methyl.

[0228] In this case, resistance to enzymatic cleavage is conferred bythe alkyl substitution on the amine.

[0229] I. Polymeric linkers are also contemplated by the presentinvention. Dextran and cyclodextran are preferred polymers useful inthis embodiment of the present invention as a result of thehydrophilicity of the polymer, which leads to favorable excretion ofconjugates containing the same. Other advantages of using dextranpolymers are that such polymers are substantially non-toxic andnon-immunogenic, that they are commercially available in a variety ofsizes and that they are easy to conjugate to other relevant molecules.Also, dextran-linked conjugates exhibit advantages when non-target sitesare accessible to dextranase, an enzyme capable of cleaving dextranpolymers into smaller units while non-target sites are not soaccessible.

[0230] Other linkers of the present invention are produced prior toconjugation to DOTA and following the reduction of the biotin carboxymoiety. These linkers of the present invention have the followinggeneral formula:

[0231] Embodiments of linkers of this aspect of the present inventioninclude the following:

[0232] J. An ether linkage as shown below may be formed in a DOTA-biotinconjugate in accordance with the procedure indicated below.

L′=—NH—CO—(CH₂)_(n)—O—

[0233] where n ranges from 1 to 5, with 1 preferred.

[0234] This linker has only one amide moiety which is bound directly tothe DOTA aniline (as in the structure of DOTA-SC-biotin). In addition,the ether linkage imparts hydrophilicity, an important factor infacilitating renal excretion.

[0235] K. An amine linker formed from reduced biotin (hydroxybiotin oraminobiotin) is shown below, with conjugates containing such a linkerformed, for example, in accordance with the procedure described inExample XXI.

L′=—NH—

[0236] This linker contains no amide moieties and the unalkylated aminemay impart favorable biodistribution properties since unalkylatedDOTA-aniline displays excellent renal clearance.

[0237] L. Substituted amine linkers, which can form conjugates viaamino-biotin intermediates, are shown below.

[0238] where R⁸ is H; —(CH₂)₂—OH or a sulfate or phosphonate derivativethereof; or

[0239] or the like; and R⁹ is a bond or —(CH₂)_(n)—CO—NH—, where nranges from 0-5 and is preferably 1 and where q is 0 or 1.

[0240] These moieties exhibit the advantages of an amide only directlyattached to DOTA-aniline and either a non-amide amine imparting apositive charge to the linker in vivo or a N-alkylated glucuronidehydrophilic group, each alternative favoring renal excretion.

[0241] M. Amino biotin may also be used as an intermediate in theproduction of conjugates linked by linkers having favorable properties,such as a thiourea-containing linker of the formula:

L′=—NH—CS—NH—

[0242] Conjugates containing this thiourea linker have the followingadvantages: no cleavable amide and a short, fairly polar linker whichfavors renal excretion.

[0243] A bis-DOTA derivative of the following formula can also be formedfrom amino-biotin.

[0244] where n ranges from 1 to 5, with 1 and 5 preferred.

[0245] This molecule offers the advantages of the previously discussedbis-DOTA derivatives with the added advantage of no cleavable amides.

[0246] Additional linkers of the present invention which are employed inthe production of conjugates characterized by a reduced biotin carboxymoiety are the following:

[0247] L=—(CH₂)₄—NH—, wherein the amine group is attached to themethylene group corresponding to the reduced biotin carboxy moiety andthe methylene chain is attached to a core carbon in the DOTA ring. Sucha linker is conveniently synthesizable from lysine.

[0248] L=—(CH₂)_(q) 13 CO—NH—, wherein q is 1 or 2, and wherein theamine group is attached to the methylene group corresponding to thereduced biotin carboxy moiety and the methylene group(s) are attached toa core carbon in the DOTA ring. This moiety is synthesizable fromamino-biotin.

[0249] The linkers set forth above are useful to produce conjugateshaving one or more of the following advantages:

[0250] bind avidin or streptavidin with the same or substantiallysimilar affinity as free biotin;

[0251] bind metal M⁺³ ions efficiently and with high kinetic stability;

[0252] are excreted primarily through the kidneys into urine;

[0253] are stable to bodily fluid amidases;

[0254] penetrate tissue rapidly and bind to pretargeted avidin orstreptavidin; and

[0255] are excreted rapidly with a whole body residence half-life ofless than about 5 hours.

[0256] Synthetic routes to an intermediate of the DOTA-biotin conjugatesdepicted above, nitrobenzyl-DOTA, have been proposed. These proposedsynthetic routes produce the intermediate compound in suboptimal yield,however. For example, Renn and Meares, “Large Scale Synthesis ofBifunctional Chelating AgentQ-(p-nitrobenzyl)-1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetraacetic acid, and the Determination of its Enantiomeric Purity by ChiralChromatography,” Bioconj. Chem., 3: 563-9, 1992, describe a nine-stepsynthesis of nitrobenzyl-DOTA, including reaction steps that eitherproceed in low yield or involve cumbersome transformations orpurifications. More specifically, the sixth step proceeds in only 26%yield, and the product must be purified by preparative HPLC.Additionally, step eight proceeds in good yield, but the processinvolves copious volumes of the coreactants.

[0257] These difficulties in steps 6-8 of the prior art synthesis areovercome in the practice of the present invention through the use of thefollowing synthetic alternative therefor.

[0258] The poor yield in step six of the prior art synthesis procedure,in which a tetra amine alcohol is converted to atetra-toluenesulfonamide toluenesulfonate as shown below, is the likelyresult of premature formation of the toluenesulfonate functionality(before all of the amine groups have been converted to theircorresponding sulfonamides.

[0259] Such a sequence of events would potentially result in unwantedintra- or inter-molecular displacement of the reactive toluene sulfonateby unprotected amine groups, thereby generating numerous undesirableside-products.

[0260] This problem is overcome in the aforementioned alternativesynthesis scheme of the present invention by reacting the tetra-aminealcohol with trifluoroacetic anhydride. Trifluoroacetates, being muchpoorer leaving groups than toluenesulfonates, are not vulnerable toanalogous side reactions. In fact, the easy hydrolysis oftrifluoroacetate groups, as reported in Greene and Wuts, “ProtectingGroups in Organic Synthesis.” John Wiley and Sons, Inc., New York, p.94, 1991., suggests that addition of methanol to the reaction mixturefollowing consumption of all amines should afford thetetra-fluoroacetamide alcohol as a substantially exclusive product.Conversion of the tetra-fluoroacetamide alcohol to the correspondingtoluenesulfonate provides a material which is expected to cyclizeanalogously to the tetra-toluenesulfonamide toluenesulfonate of theprior art. The cyclic tetra-amide product of the cyclization of thetoluenesulfonate of tetra-fluoroacetamide alcohol, in methanolic sodiumhydroxide at 15-25° C. for 1 hour, should afford nitro-benzyl-DOTA as asubstantially exclusive product. As a result, the use oftrifluoracetamide protecting groups circumvents the difficultiesassociated with cleavage of the very stable toluenesulfonaamideprotecting group, which involves heating with a large excess of sulfuricacid followed by neutralization with copious volumes of bariumhydroxide.

[0261] Another alternative route to nitro-benzyl-DOTA is shown below.

[0262] This alternative procedure involves the cyclizaton ofp-nitrophenylalanyltriglycine using a coupling agent, such asdiethylycyanophosphate, to give the cyclic tetraamide. Subsequent boranereduction provides 2-(p-nitrobenzyl)-1,4,7,10-tetraazacyclododecane, acommon precursor used in published routes to DOTA including the Renn andMeares article referenced above. This alternative procedure of thepresent invention offers a synthetic pathway that is considerablyshorter than the prior art Renn and Meares route, requiring two ratherthan four steps betwe n p-nitrophenylalanyltriglycine to the tetraamine.The procedure of the present invention also avoids the use of tosylamino protecting groups, which were prepared in low yield and requiredstringent conditions for removal. Also, the procedure of the presentinvention poses advantages over the route published by Gansow et al.,U.S. Pat. No. 4,923,985, because the crucial cyclization step isintramolecular rather than intermolecular. Intramolecular reactionstypically proceed in higher yield and do not require high dilutiontechniques necessary for successful intermolecular reactions.

[0263] In order to more effectively deliver a therapeutic or diagnosticdose of radiation to a target site, the radionuclide is preferablyretained at the tumor cell surface. Loss of targeted radiation occurs asa consequence of metabolic degradation mediated by metabolically activetarget cell types, such as tumor or liver cells.

[0264] Preferable agents and protocols within the present invention aretherefore characterized by prolonged residence of radionuclide at thetarget cell site to which the radionuclide has localized and improvedradiation absorbed dose deposition at that target cell site, withdecreased targeted radioactivity loss resulting from metabolism.Radionuclides that are particularly amenable to the practice of thisaspect of the present invention are rhenium, iodine and like “non +3charged” radiometals which exist in chemical forms that easily crosscell membranes and are not, therefore, inherently retained by cells. Incontrast, radionuclides having a +3 charge, such as In-111, Y-90, Lu-177and Ga-67, exhibit natural target cell retention as a result of theircontainment in high charge density chelates.

[0265] Evidence exists that streptavidin is resistant to metabolicdegradation. Consequently, radionuclides bound directly or indirectly tostreptavidin, rather than, for example, directly to the targetingmoiety, are retained at target cell sites for extended periods of time,as described below in Examples XIV and XV. Streptavidin-associatedradionuclides can be administered in pretargeting protocols or injecteddirectly into lesions.

[0266] In addition, streptavidin-associated radionuclides (e.g.,streptavidin-radionuclide and streptavidin-biotin-radionuclide) may beadministered as such (in pretargeting protocols) or as conjugatesincorporating targeting moieties (intralesional injection andpretargeting protocols) specific for stable target cell surface antigens(such as NR-LU-10 antibody, L6, anti-CEA antibodies or the like) ortarget cell internalizing antigens (such as anti-HER2^(neu);anti-epidermal growth factor; anti-Lewis Y, including B-1, B-3, BR-64,BR-96 and the like; or the like) to target the streptavidin to theappropriate target cell population.

[0267] Streptavidin associated-radionuclides are amenable, for example,to intralesional injection of ovarian cancer lesions studded on theperitoneum and accessible via laparotomy. Another example of anintralesional injection aspect of the present invention involveshepatoma or liver cancer, preferably using a terminalgalactose-streptavidin derivative to bind a radionuclide.

[0268] Moreover, high molecular weight carriers, such as biodegradableparticles, dextran, albumin or the like, may be employed (e.g.,conjugated to streptavidin) to limit leakage of the administeredstreptavidin from the injection site. Alternatively, such carriers arebiotinylated, thereby constituting suitable targets or carriers forradionuclide-streptavidin molecules.

[0269] The use of streptavidin-associated radionuclides in intralesionalinjection protocols provides the following advantages:

[0270] less radionuclide is used to better advantage, because thetherapeutic efficacy of the administered radionuclide is improved as aresult of retention at the target cell site;

[0271] microdiffusion from the injection site results in expansion ofthe field of radiation deposition;

[0272] minimized toxicity and higher dose rate radiation are achieved;

[0273] combination with modalities exhibiting disparate toxicityprofiles“may be useful;

[0274] target sites are imageable post-injection to allow dosimetrydeterminations to be made;

[0275] biodegradable (i.e., not requiring removal) retentionmoiety-carrier molecules can be utilized; and

[0276] repeated doses can be injected, because local administrationwithout systemic distribution minimizes antiglobulin response.

[0277] The use of streptavidin-associated radionuclides in pretargetingprotocols provides the following advantages:

[0278] less radionuclide is used to better advantage, because thetherapeutic efficacy of the administered radionuclide is improved as aresult of retention at the target cell site;

[0279] target sites are imageable post-injection to allow dosimetrydeterminations to be made;

[0280] minimized toxicity and higher dose rate radiation are achieved;and

[0281] combination with modalities exhibiting disparate toxicityprofiles may be useful.

[0282] In addition, the target cell retention-enhancing aspect of thepresent invention is applicable to a hybrid pretargeting/intralesionalinjection protocol. For example, targeting moiety-biotin conjugate isadministered and an intralesional injection of streptavidin followsafter a time sufficient to permit localization of the targetingmoiety-biotin conjugate to target cell sites of reasonably determinablelocation. Next, a radionuclide-biotin molecule is administered, whereinthis administration is conducted by intralesional, intravenous or otherconvenient route.

[0283] Monovalent antibody fragment-streptavidin conjugate may be usedto pretarget streptavidin, preferably in additional embodiments of thetwo-step aspect of the present invention. Exemplary monovalent antibodyfragments useful in these embodiments are Fv, Fab, Fab′ and the like.Monovalent antibody fragments, typically exhibiting a molecular weightranging from about 25 kD (Fv) to about 50 kD (Fab, Fab′), are smallerthan whole antibody and, therefore, are generally capable of greatertarget site penetration. Moreover, monovalent binding can result in lessbinding carrier restriction at the target surface (occurring during useof bivalent antibodies, which bind strongly and adhere to target cellsites thereby creating a barrier to further egress into sublayers oftarget tissue), thereby improving the homogeneity of targeting.

[0284] In addition, smaller molecules are more rapidly cleared from arecipient, thereby decreasing the immunogenicity of the administeredsmall molecule conjugate. A lower percentage of the administered dose ofa monovalent fragment conjugate localizes to target in comparison to awhole antibody conjugate. The decreased immunogenicity may permit agreater initial dose of the monovalent fragment conjugate to beadministered, however.

[0285] A multivalent, with respect to ligand, moiety is preferably thenadministered. This moiety also has one or more radionuclides associatedtherewith. As a result, the multivalent moiety serves as both a clearingagent for circulating anti-ligand-containing conjugate (throughcross-linking or aggregation of conjugate) and as a therapeutic agentwhen associated with target bound conjugate. In contrast to theinternalization caused by cross-linking described above, cross-linkingat the tumor cell surface stabilizes the monovalent fragment-anti-ligandmolecule and, therefore, enhances target retention, under appropriateconditions of antigen density at the target cell. In addition,monovalent antibody fragments generally do not internalize as dobivalent or whole antibodies. The difficulty in internalizing monovalentantibodies permits cross-linking by a monovalent moiety which serves tostabilize the bound monovalent antibody through multipoint binding. Thistwo-step protocol of the present invention has greater flexibility withrespect to dosing, because the decreased fragment immunogenicity allowsmore streptavidin-containing conjugate, for example, to be administered,and the simultaneous clearance and therapeutic delivery removes thenecessity of a separate controlled clearing step.

[0286] Another embodiment of the pretargeting methodologies of thepresent invention involves the route of administration of the ligand- oranti-ligand-active agents. In these embodiments of the presentinvention, the active agent-ligand (e.g., radiolabeled biotin) or-anti-ligand is administered intraarterially using an artery supplyingtissue that contains the target. In the radiolabeled biotin example, thehigh extraction efficiency provided by avidin-biotin interactionfacilitates delivery of very high radioactivity levels to the targetcells, provided the radioactivity specific activity levels are high. Thelimit to the amount of radioactivity delivered therefore becomes thebiotin binding capacity at the target (i.e., the amount of antibody atthe target and the avidin equivalent attached thereto).

[0287] For these embodiments of the pretargeting methods of the presentinvention, particle emitting therapeutic radionuclides resulting fromtransmutation processes (without non-radioactive carrier forms present)are preferred. Exemplary radionuclides include Y-90, Re-188, At-211,Bi-212 and the like. Other reactor-produced radionuclides are useful inthe practice of these embodiments of the present invention, if they areable to bind in amounts delivering a therapeutically effective amount ofradiation to the target. A therapeutically effective amount of radiationranges from about 1500 to about 10,000 cGy depending upon severalfactors known to nuclear medicine practitioners.

[0288] Intraarterial administration pretargeting can be applied totargets present in organs or tissues for which supply arteries areaccessible. Exemplary applications for intraarterial delivery aspects ofthe pretargeting methods of the present invention include treatment ofliver tumors through hepatic artery administration, brain primary tumorsand metastases through carotid artery administration, lung carcinomasthrough bronchial artery administration and kidney carcinomas throughrenal artery administration. Intraarterial administration pretargetingcan be conducted using chemotherapeutic drug, toxin and anti-tumoractive agents as discussed below. High potency drugs, lymphokines, suchas IL-2 and tumor necrosis factor, drug/lymphokine-carrier-biotinmolecules, biotinylated drugs/lymphokines, anddrug/lymphokine/toxin-loaded, biotin-derivitized liposomes are exemplaryof active agents and/or dosage forms useful for the delivery thereof inthe practice of this embodiment of the present invention.

[0289] A problem associated with solid tumor target sites, for example,is penetration of the therapeutic agent into the active site. Ifhomogeneous penetration of such target sites is achieved, more effectivetherapy is possible. One method that may be employed to enhance targetsite penetration of ligand or anti-ligand conjugated to a targetingagent or anti-ligand or ligand conjugated to a therapeutic moiety is theadministration of a permeability enhancing moiety that induces orpromotes vascular leakiness, disrupts cell-to-cell associations orsimilarly facilitates more homogeneous delivery of an administeredconjugate to a target site characterized by a three dimensional cellulararray. Preferably, permeability enhancing moieties of the presentinvention achieve their effects rapidly (e.g., within from about 30seconds to about 1 hour following administration).

[0290] One example of this aspect of the present invention involvesanti-ligand-targeting moiety conjugate administration (e.g.,systemically, intra-arterially, locally via catheter or the like)followed, for example, by intravascular perfusion catheteradministration of a permeability enhancing moiety (e.g., an agent thatinduces gaps in the endothelium of venules through action on thepostcapillary venules such as histamine, serotonin or bradykinin; orthrough action on the-entire capillary bed such as bacterial endotoxins;or the like) which disrupts the target three dimensional structure andinduces microvascular leakiness, thereby permitting simultaneously orsubsequently administered ligand-therapeutic agent conjugates to achievea higher interstitial concentration at the target site. One method toachieve site specificity for the therapeutic agent conjugateadministration is to administer that agent via catheter. The optimalroute of administration for each administered component will be dictatedby the recipient's physiological condition and the specific treatmentselected therefor by an experienced medical practitioner. Optionally, aclearing agent may be administered prior or subsequent to thepermeability enhancing moiety, with prior administration preferred.

[0291] The use of this aspect of the present invention also facilitatesthe delivery of higher molecular weight therapeutic agent-bearingconjugates to target sites characterized by a three dimensional cellulararray. For example, one or more ligand or anti-ligand molecules as wellas a plurality of therapeutic agent molecules may be conjugated to apolymer to form an entity of the following formula:

Therapeutic Agent₁₋₃₀-Polymer-Ligand or Anti-ligand₁₋₁₀

[0292] A specific embodiment of this -aspect of the present invention,useful particularly for target sites characterized by an accessible (foradministration purposes) arterial supply as discussed herein, involvesintraarterial administration of permeability enhancing agent followed bysuch administration of therapeutic agent-polymer-ligand or -anti-ligandconjugate. An exemplary conjugate suitable for such delivery is abiotinylated derivative of the hydroxy-propylmethacrylate-adriamycinconjugate discussed by R. Duncan and J. Kopecek, Advances in PolymerScience, 57: 52-101 (1984) and R. Duncan, Anticancer Drugs, 3: 175-210(1992).

[0293] Alternatively, the permeability enhancing moiety may beadministered prior to administration of the targeting moiety-ligand or-anti-ligand conjugate to facilitate more homogenous targeting of thatadministered conjugate which serves as a receptor for subsequentlyadministered anti-ligand- or ligand-therapeutic agent conjugate.Additionally, permeability enhancing moieties may also be employed inthree-step pretargeting protocols of the present invention.

[0294] Preferred permeability enhancing moieties of the presentinvention induce gaps in the endothelium of venules through “specific”or “non-specific” interaction. For the purposes of this discussion,non-specific permeability enhancing moieties are those that act upon theentire capillary bed. Exemplary permeability enhancing moieties of thenon-specific type are bacterial endotoxins, metabolic inhibitors, drugsthat alter microfilaments or microtubules and the like. Specificpermeability enhancing moieties act on a portion of the capillary bed(e.g., on the postcapillary venules, capillaries or the like). Exemplarypermeability enhancing agents of the specific type are histamine,serotonin, bradykinin, and the like. Other permeability enhancingmoieties are mannitol, tumor necrosis factor, nitric oxide,prostaglandin E2, leukotriene, leukokinin and the like.

[0295] Alternatively, enhanced delivery of a diagnostic or therapeuticsubstance, for example, into a target tumor mass, can be achieved bysystemically administering a conjugate containing a targeting moiety, amember of a ligand/anti-ligand binding pair and an amount of apermeability enhancing agent sufficient to achieve disruption ofcell-to-cell association within the tumor mass. Such a permeabilityenhancing moiety may also be locally administered, for example, in thepractice of the local or intraarterial administration protocolsdescribed above.

[0296] Exemplary permeability enhancing moieties of the presentinvention include any substance capable of disrupting cell-to-cellassociation within a three dimensional target cell array. Some preferredmoieties include trichothecenes, which are small molecule proteinsynthesis inhibitors, ionophores, membrane-active compounds(particularly inhibitors of microtubule or microfilament polymerization,polymyxins and polyene antibiotics), cyclic toxin peptides,cytochalasins and combinations thereof. Verrucarin A, microcystin (fromMicrocystin aeruginosa), cydloheximide and puromycin are particularlypreferred permeability enhancing moieties.

[0297] Moieties possessing a macrocyclic ring, a functional epoxidegroup or both are also preferred within the present invention. However,cytotoxic compounds that are devoid of macrocyclic rings may also besuitable for use herein.

[0298] Small permeability enhancing moieties having a molecular weightless than or equal to about 2,000 daltons, and particularly those havinga molecular weight less than or equal to 1,000 daltons, are alsopreferred. Such small permeability enhancing moieties are able to passfreely between adjacent cells whose cytoplasms are coupled via gapjunctional complexes.

[0299] In a systemic administration embodiment, a permeability enhancingmoiety having a molecular weight of about 1,000 daltons or less isconjugated to a targeting moiety (preferably also having a ligand or ananti-ligand bound thereto) through a selectively cleavable covalentlinkage. In this embodiment, after delivery of the conjugate to a targetcell, the permeability enhancing moiety is released at the target cell,and is then distributed to adjacent cells through gap junctions. Freediffusion of the released moiety will increase the efficiency, forexample, of tumor mass disruption.

[0300] For some permeability enhancing moieties, it may be advantageousto conjugate multiple moieties to a carrier molecule through aselectively cleavable covalent linkage. As used herein, “carriermolecule” includes, but is not limited to, a large protein or polymercapable of binding many permeability enhancing moieties to a singletargeting moiety. Preferred carrier molecules include albumin, dextran,hydroxy-propylmethacrylamide, poly-L-lysine and polyglutamate, all ofwhich have a molecular weight in excess of 5,000 daltons. Carriermolecules may also be produced through chemical syntheses or recombinantDNA technology. A carrier molecule generally is derivatized with smallselectively cleavable linking groups, which enable binding of one ormore permeability enhancing moieties to the carrier. The “permeabilityenhancing moiety loaded” carrier is then either locally orintraarterially administered or systemically administered after beingcovalently attached to a targeting moiety (directly or through alinker).

[0301] Alternatively, specific target site vascular permeability isachievable by administering an effective amount of a permeabilityenhancing moiety which is a target cell inflammatory response mediator.A preferred permeability enhancing moiety of this type is a substancethat is capable of localizing at a target site and capable of mediatingcomplement-dependent inflammation at the target site within a patient.Such moieties are recognized by their ability to mediatecomplement-dependent cytotoxicity in vitro with the recipient's serum.Permeability enhancing moieties may be monoclonal antibodies, polyclonalantibodies, chimeric antibodies, human or humanized antibodies or othermoiety having the capabilities described above.

[0302] Permeability enhancing moieties of this type may also beadministered as a conjugate with a vasoactive complement-derivedpeptide, such as a peptide derived from C2. Complement-derived peptidesare linked to the permeability enhancing moieties in a manner permittingrelease of the complement-derived peptide at the target site, such asthrough a labile linkage (i.e., a Schiff base linkage).

[0303] Preferably, a subset of antibodies, such as mouse antibodies,human/mouse chimeric antibodies or humanized antibodies are used toinduce target site-specific increases in vascular permeability. Thesemonoclonal antibodies mediate target cell lysis and/or target cellinflammatory responses in vitro which contribute to target site-specificvascular permeability increases. Both direct and indirect action ontarget cells may be accomplished by mediation of complement-dependentcytotoxicity. The ability to mediate complement-dependent cytotoxicityin vitro is indicative of the ability to mediate complement-dependentinflammation in vivo.

[0304] The invention exploits one attribute of antibodies—possession ofisotype- and/or subclass-specific functions. For example, murine IgG₃and human/mouse chimeric antibodies having human IgG₁ or IgG₃ constantportions are generally considered superior to other immunoglobulins ofmurine or murine/human nature for mediating antibody-dependentcell-mediated inflammation (ADCC) or complement-dependent cellulartoxicity. Antibodies or other moieties effective in mediatingtarget-site complement-dependent cytotoxicity must be capable ofutilizing the serum complement native to the ultimate recipient havingconfirmed or potential target sites. Consequently, human antibodies areexpected to be capable of utilizing human serum. Antibodies of othertypes are therefore tested for this ability when administration to humanrecipients is contemplated.

[0305] Exemplary moieties known to mediate dependent cytotoxicity areNR—CO-04, a murine IgG₃ antibody directed to colon carcinoma; NR-LU-13,a murine/human chimeric antibody featuring a human constant region andmurine variable region of NR-LU-10, an antibody directed to a 37-40kilodalton pancarcinoma glycoprotein; NR-LU-03, an IgG₃ antibodygenerated by immune complex immunization with NR-LU-10; R24, an IgG₃antibody directed against GD3 disclosed by Houghton et al., Proc. Natl.Acad. Sci (USA), 82:1242-1246, 1985; antibody 3F8, a mouse IgG₃ directedagainst GD2 disclosed by Munn et al., Cancer Res., 47:6600-6605, 1987and Cheung et al., J. Clin. Invest., 81:1122-1128, 1988; and the like.

[0306] Some polymers useful in the practice of this permeabilityenhancing aspect of the present invention serve solely as a carrier formultiple therapeutic agents. Preferred polymers also direct thebiodistribution of the ligand or anti-ligand and therapeutic agents towhich the polymer is bound to renal rather than, for example,hepatobiliary excretion. For many administered therapeutic agents (e.g.,radionuclides), renal excretion is preferred, especially for therapeuticprotocols.

[0307] Exemplary polymers for use in the practice of the presentinvention are excreted or which metabolites thereof are excreted througha renal pathway when administered to a mammalian recipient and that iscapable of covalently or non-covalently binding to one or more drugs,anti-tumor agents, peptides, chelates, ligands, anti-ligands or othersmall molecules and imposing a renal route of excretion upon theassociated molecule(s). Such polymers include polar molecules having amolecular weight ranging from about 3 kD to about 70 kD).

[0308] Exemplary polymers useful in permeability enhancing aspects ofthe present invention are dextran; dextran derivatives includingcarboxymethyl dextran, anionic or polar derivatives thereof such ascarboxymethyl-dextran, 3-mercapto-2-hydroxypropyl dextran and the like;hyaluronic acid, inulin, carboxymethyl cellulose;hydroxy-propylmethacrylamide (HPMA) polymers; succinylated polylysine;polyaspartate; polyglutamate; polyethyleneglycol (PEG); and the like.Dextran polymers are described herein as the prototypical polymers foruse in the permeability enhancing aspects of the invention.

[0309] For use in the present invention, dextran polymers preferablyrange between about 5 and about 15 kD in size, although larger moietiesmay also be used. When larger (from about 40 to about 70 kD) polymersare employed in accordance with the present invention, the rate ofclearance of the conjugate from a recipient is slowed, but the renalpathway for excretion is maintained. In this manner, the moieties boundto the polymer exhibit increased bioavailability as a result of theincreased circulation time thereof. Such bound molecules remain directedto renal excretion, however.

[0310] Exemplary dextran-containing conjugates of the present inventionare dextran-biotin conjugates. Chelate-biotin-dextran conjugates of thepresent invention are formed, for example, from oxidized dextran byconjugating biocytin hydrazide thereto followed by reaction with achelate active ester, such as a N-hydroxy succinimidyl ester, atetrafluorophenyl ester and the like. Alternatively,chelate-biotin-dextran can be formed by reacting dextran hydrazide withthe carboxy terminus of a chelate-biocytin conjugate. Preferably, thecarboxy terminus has been derivitized to form an active ester, such asan N-hydroxysuccinimidyl ester or the like an alternative protocol ofgeneral applicability to different polymers is discussed in ExampleXVII.

[0311] Biotin-dextran, lysine fixable (BDLF, available from SigmaChemical Company, St. Louis, Mo.) is a preferred polymer-ligand for usein the practice of the present invention.

[0312] Active agents can be bound to dextran polymers characterized byshort term serum stable linkages disposed between monomeric, dimeric,trimeric or other convenient unit thereof. Such conjugates are alsopreferably large (exhibiting a molecular weight ranging from about 40 toabout 70 kD). One advantage of the use of such polymers is that themolecules affixed thereto will exhibit an increased circulation time aswell as decreased liver uptake. More specifically, the circulation timeof the bound molecules is dictated by the maintenance of polymericstructural integrity in vivo. Depolymerization releases boundmolecule-monomer, -dimer, -trimer or like moieties that are themselvesrapidly cleared from the recipient's circulation, preferably via therenal pathway.

[0313] Depolymerization may be controlled in any convenient mannertherefor, including, for example, the following methods:

[0314] Use of linkages between dextran units containing chemical groupsthat are stable in serum for a period of time sufficient to provide anappropriate circulation time to the small molecules bound thereto (e.g.,1 to 3 hours for active agent in both the pretargeting and targeted,direct labeled protocols); or

[0315] Use of linkages between dextran units that are enzymaticallycleaved upon administration of enzyme after the passage of anappropriate amount of bound molecule circulation time.

[0316] In the first approach, groups such as esters, acetals,disulfides, thioacetals or the like are employed. For example, an esterlinkage, having a serum stability of 1-3 hours such as phenyl oractivated phenyl or phthalyl (e.g., chloro-substituted,fluoro-substituted, multi-halogen-substituted, nitro-substituted or thelike), may be employed to attach dextran polymer units. Such a linkageis stable in serum for a time sufficient to facilitate localization ofthe molecules bound to the polymer to the target site.

[0317] The second approach includes the use of dextran units susceptibleto cleavage by an administrable enzyme that is not found in largeamounts in human serum. Exemplary enzymes useful in the practice of thisaspect of the present invention include dextranase, alpha-amylase,pullulanase (a bacterial alpha-1,6-polysaccharidase) and the like. Theenzymes are administered by any convenient route in any convenientdosage form therefor. Such enzymes are optionally conjugated or formedas fusion proteins with long circulating proteins, including albumin,immunoglobulins or portions thereof, and the like. In this manner, theadministered enzymes remain in circulation for a time sufficient toeffect depolymerization of the polymer.

[0318] As discussed previously, cross-linking of moieties bound to atarget cell surface results in internalization of those bound moietiesby the cell. This cross-linking phenomena can be exploited in severalways using the 2-step and 3-step pretargeting protocols of the presentinvention. Three exemplary protocols of thecross-linking/internalization aspect of the present invention using theexemplary biotin/avidin ligand/anti-ligand pair can be described asfollows:

[0319] 1) (two-step) administer targeting moiety-biotin-therapeuticagent conjugate (or biotin-targeting moiety-therapeutic agentconjugate); and

[0320] administer avidin or streptavidin to cross-link the previouslylocalized conjugate. A variation of this protocol involvesadministration of targeting moiety-biotin conjugate followed byadministration of therapeutic agent-avidin or -streptavidin conjugate.

[0321] 2) (two-step) administer avidin- or streptavidin-targetingmoiety-therapeutic agent conjugate (or therapeutic agent-avidin- orstreptavidin-targeting moiety conjugate; or a therapeuticagent-containing conjugate that also incorporates Antibody-(avidin orstreptavidin) ₂ or (Antibody)₂-avidin or -streptavidin); and

[0322] administer multiple biotin-polymer conjugate to cross-link thepreviously localized conjugate. A variation of this protocol involvesadministration of a targeting moiety-avidin or -streptavidin conjugate,Antibody-(avidin or streptavidin)₂ conjugate or (Antibody)₂-avidin or-streptavidin conjugate followed by administration of multiplebiotin-polymer therapeutic agent conjugate.

[0323] 3) (three-step) administer targeting moiety-biotin conjugate;

[0324] administer avidin or streptavidin;

[0325] administer multiple biotin-polymer-therapeutic agent conjugate toeither cross-link the previously localized avidin or streptavidin or tobe internalized into the cell as a result of the avidin or streptavidincross-linking of previously localized biotin-containing conjugate.

[0326] These cross-linking/internalization embodiments of the presentinvention are exemplified below with reference to the biotin/avidinligand/anti-ligand pair, monoclonal antibody targeting moieties andtrichothecene therapeutic agents; however, the embodiments are amenableto use with other administered components.

[0327] Exemplary simple trichothecenes useful in the practice of thisand other aspects of the present invention are as follows:

[0328] wherein:

[0329] R₁ is H, OH,

[0330] SH or L₁, L₂, L₃, or L₄;

[0331] R₂ is H, OH, or

[0332] R₃ is H, OH, or

[0333] R₄ is H, or forms an epoxide group with R₅;

[0334] R₅ is H, OH,

[0335] SH, OCH₃. SCH₃, NH₂, NHCH₃, N(CH₃)₂, L₃, L₄, or forms an epoxidegroup with R₄; and

[0336] R₆ is H, OH, SH, OCH₃, SCH₃, NH₂, NHCH₃, N(CH₃)₂, L₃, or L₄

[0337] provided that at least one of R₁, R₅ and R₆ is L₁, L₂, L₃, or L₄and further provided that R₅ and R₆ are not L₁ or L₂ and wherein

[0338] and wherein

[0339] Y is O or NH,

[0340] R₇ or R_(7′) is H or CH₃,

[0341] R₈ or R_(8′) is H or OH,

[0342] n is one to ten; and

[0343] n′ is zero to ten.

[0344] Preferred simple trichothecenes are compounds where R₂ and R₃ areboth

[0345] and R₄ and R₅ are both H. Other preferred trichothecenes arecharacterized as follows:

[0346] (1) R₁ is L₁ or L₂ and Y is NH, R₇ and R₇, are independentlyeither H or CH₃, R₈ and R₈, are independently either H or OH, and n′ iszero to ten; further provided that R₂ and R₃ are both

[0347] R₄ is H; and R₅ and R₆ are selected from the group consisting ofH, OH, SH, OCH₃, SCH₃, NH₂, NHCH₃, N(CH₃)₂;

[0348] (2) R₁ is H, OH or SH; and R₅ and R₆ are selected from the groupconsisting of H, OH, SH, OCH₃, SCH₃, NH₂, NHCH₃, N(CH₃)₂, or L₃, whereinR₇ and R₇, are independently either H or CH₃ and n is one to ten;

[0349] (3) R₁ is H, OH or SH; and R₅ or R₆ is selected from the groupconsisting of H, OH, SH, OCH₃, SCH₃, NH₂, NHCH₃, N(CH₃)₂, or L₄, whereinn is one to ten, and R₇ and R_(7′) are independently either H or CH₃;

[0350] (4) R₁ is L₃ or L₄ wherein R₇ and R_(7′) and independently eitherH or CH₃ and n is one to ten; and R₅ and R₆ are selected from the groupconsisting of H, OH, SH, OCH₃, SCH₃, NH₂, NHCH₃, N(CH₃)₂.

[0351] A prototypical simple trichothecene is anguidine or the 3-ketoderivative thereof which may be used in protocol number 1 set forthabove as, for example, Biotin-NR-LU-10 monoclonalantibody-S—S—(CH₂)₂—CONH—N=(3-keto anguidine). Similarly, this simpletrichothecene may be utilized in the protocols numbered 2 and 3 aboveas, for example, (Biotin)₁₈₋₂₀-Dextran-anguidine. Within the context ofthe above presented structures a prototypical simple trichothecene ischaracterized as follows: R₁ is L₁ and Y is NH, R₇ and R₇, are both H,and n′ is zero; R₄, R₅, R₆ are H; and R₂ and R₃ are both

[0352] Exemplary macrocyclic trichothecenes useful in the practice ofthis and other aspects of the present invention are as follows:

[0353] wherein R′ is selected from the group consisting of

[0354] provided at least one of R₁, W and Z is L₁, L₂, L₃, or L₄, andfurther provided W and Z are not both L₁, L₂, L₃, or L₄ wherein

[0355] and wherein

[0356] Y is O or NH,

[0357] R₇ or R_(7′) is H or CH₃,

[0358] R₈ or R_(8′) is H or OH,

[0359] n is one to ten; and

[0360] n′ is zero to ten;

[0361] and further provided that W can be an epoxide group between 2′and 3′ and still further provided that W and Z can be independentlyeither H, OH, or SH when W and Z are not L₁, L₂, L₃, or L₄; R₁ is H, OH,or SH when R¹ is not L₁, L₂, L₃, or L₄; and R₂ and R₃ are selected fromthe group consisting of, H, OH, SH, OCH₃, SCH₃, NH₂, NHCH₃, N(CH₃)₂, L₃or L₄.

[0362] Preferred macrocyclic trichothecenes are compounds wherein R′ is

[0363] Such preferred compounds are derivatives of Roridin A. Morepreferred macrocyclic trichothecenes of this type are characterized asfollows:

[0364] (1) R₁ is L₁, L₂, L₃, or L₄ wherein

[0365] Y is O or NH,

[0366] R₇ and R_(7′) are independently either H or CH₃,

[0367] R₈ and R_(8′) are independently either H or OH,

[0368] n is one to ten,

[0369] n′ is zero to ten;

[0370] when R₂ and R₃ are selected from the group consisting of, H, OH,SH, OCH₃, SCH₃, NH₂, NHCH₃, N(CH₃)₂; and W and Z are independentlyeither H, OH, or SH;

[0371] (2) R₁ and W are independently either H, OH, or SH; R₂ and R₃ areselected from the group consisting of, H, OH, SH, OCH₃, SCH₃, NH₂,NHCH₃, N(CH₃)₂; and Z is L₁, L₂, L₃, or L₄ and wherein

[0372] Y is O or NH,

[0373] R7 or R_(7′ is H or CH) ₃,

[0374] R₈ or R_(8′) is H or OH,

[0375] n is one to ten; and

[0376] n′ is zero to ten;

[0377] (3) R₁ and Z are H, SH, or OH; R₂ and R₃ are selected from thegroup consisting of, H, OH, SH, OCH₃, SCH₃, NH₂, NHCH₃, N(CH₃)₂; and Wis either L₁, L₂, L₃, or L₄ and wherein

[0378] Y is O or NH,

[0379] R₇ or R_(7′) is H or CH₃.

[0380] R₈ or R_(8′) is H or OH,

[0381] n is one to ten, and

[0382] n′ is zero to ten; and

[0383] (4) R₁, W and Z are independently either SH or OH; R₂ and R₃ areselected from the group consisting of H, OH, SH, OCH₃, SCH₃, NH₂, NHCH₃,N(CH₃)₂, L₃, or L₄ wherein

[0384] R₇ and R₇, are either H or CH₃, and

[0385] n is one to ten;

[0386] provided that R₂ and R₃ are not both simultaneously L₃ or L₄.

[0387] Prototypical macrocyclic trichothecenes are Roridin A andderivatives thereof such as the 2′-oxo or the 13′-oxo derivatives. Thesetrichothecenes may be used in protocol number I set forth above as, forexample, Biotin-NR-LU-10 monoclonal antibody-S—S—(CH₂)₂—CONH—N=(2′-oxoRoridin A), Biotin-NR-LU-10 monoclonalantibody-S—S—(CH₂)₂—CONH—N=(13′-oxo Roridin A), 2′-oxo-RoridinA-S—S—(CH₂)₂—CONH-lysine-NR-LU-10 monoclonal antibody-biotin and thelike. Similarly, these macrocyclic trichothecenes may be utilized in theprotocols numbered 2 and 3 above as, for example, (Biotin)₁₈₋₂₀-Dextran-2′-oxo Roridin A.

[0388] Prototypical Roridin A derivative macrocyclic trichothecenes maybe characterized as follows: R₁ and R₂ are both H; R₃ is CH₃; W is L₁, Yis NH, R₇ and R_(7′) are both H, n′ is zero; and Z is OH;

[0389] R₁ and R₂ are both H; R₃ is CH₃; W is

[0390] and Z is L₁, Y is NH, R₇ and R_(7′) are both H, n′ is zero; and

[0391] R₁ and R₂ are both H; R₃ is CH₃; W is L₃, R₇ and R₇, are both H,n is one; and Z is OH.

[0392] Acid labile linker technology, e.g., hydrazone linkers,facilitate release of therapeutic agent in target cell endosomes andlysosomes (pH 3.5-5.5) where the released agent can exert itstherapeutic effect (e.g., inhibition of protein synthesis). Disulfidelinkages also promote release of therapeutic agent in endosomes andlysosomes of the target cells.

[0393] Exemplary protocols for trichothecene-linker preparation arediscussed in Example XVIII. Exemplary protocols fortrichothecene-antibody conjugation are set forth in Example XIX (A andB). Also, an exemplary protocol for the preparation oftrichothecene-polymer conjugates is set forth in Example XIX (C).

[0394] In embodiments of-the present invention employing radionuclidetherapeutic agents, the rapid clearance of nontargeted therapeutic agentdecreases the exposure of non-target organs, such as bone marrow, to thetherapeutic agent. Consequently, higher doses of radiation can beadministered absent dose limiting bone marrow toxicity. In addition,pretargeting methods of the present invention optionally includeadministration of short duration bone marrow protecting agents, such asWR 2721. As a result, even higher doses of radiation can be given,absent dose -limiting bone marrow toxicity.

[0395] While the pretargeting protocols set forth above have beendescribed primarily in combination with delivery of a radionuclidediagnostic or therapeutic moiety, the protocols are amenable to use fordelivery of other moieties, including anti-tumor agents,chemotherapeutic drugs and the like. For example, most naturallyoccurring and recombinant cytokines have short in vivo half lives. Thischaracteristic limits the clinical effectiveness of these molecules,because near toxic doses are often required. Dose-limiting toxicities inhumans have been observed upon high dose IL-2 or tumor necrosis factoradministrations, for example.

[0396] A protocol, such as administration of streptavidin-targetingmoiety conjugate followed by administration of biotinylated cytokine, isalso contemplated by the present invention. Such pretargeting ofanti-ligand serves to improve the performance of cytokine therapeuticsby increasing the amount of cytokine localized to target cells.

[0397] Streptavidin-antibody conjugates generally exhibitpharmacokinetics similar to the native antibody and localize well totarget cells, depending upon their construction. Biotinylated cytokinesretain a short in vivo half-life; however, cytokine may be localized tothe target as a result of the affinity of biotin for avidin. Inaddition, biotin-avidin experience a pH-dependent dissociation whichoccurs at a slow rate, thereby permitting a relatively constant,sustained release of cytokine at the target site over time. Also,cytokines complexed to target cells through biotin-avidin associationare available for extraction and internalization by cells involved incellular-mediated cytotoxicity.

[0398] A pre-formed antibody-streptavidin-biotin-cytokine preparationmay also be employed in the practice of these methods of the presentinvention. In addition, a three-step protocol of the present inventionmay also be employed to deliver a cytokine, such as IL-2, to a targetsite.

[0399] Other anti-tumor agents that may be delivered in accordance withthe pretargeting techniques of the present invention are selecting,including L-selectin, P-selectin and E-selectin. The presence ofcytokines stimulates cells, such as endothelial cells, to expressselectins on the surfaces thereof. Selectins bind to white blood cellsand aid in delivering white blood cells where they are needed.Consequently, a protocol, such as administration of streptavidin- oravidin-targeting moiety conjugate followed by administration ofbiotinylated selectins, is also contemplated by the present invention.Such pretargeting of anti-ligand serves to improve the performance ofselectin therapeutics by increasing the amount of selectin localized totarget cells. In this manner, the necessity of cytokine induction ofselectin expression is obviated by the localization and retention ofselectin at a target cell population. A three-step protocol may-also beemployed to deliver selectins to a target site.

[0400] Chemotherapeutic drugs also generally exhibit short in vivohalf-lives at a therapeutically effective dose. Consequently, anotherexample of a protocol of the present invention includes administrationof avidin-targeting moiety conjugate followed by administration of abiotin-chemotherapeutic drug conjugate or complex, such as adrug-carrier-biotin complex. A three-step protocol of the presentinvention may also be employed to deliver a chemotherapeutic drug, suchas methotrexate, adriamycin, high potency adriamycin analogs,trichothecenes, potent enediynes, such as esperamycins andcalicheamycins, cytoxan, vinca alkaloids, actinamycin D, taxol, taxotereor the like to a target site.

[0401] An additional aspect of the present invention is a method oferadicating target cells within a mammal, involving the steps of:

[0402] 1) extracting effector cells (or precursors thereof) from themammal;

[0403] 2) cultivating the extracted effector cells in vitro with both aneffector cell growth factor and a first immunogenic molecule, therebyproducing sensitized effector cells recognizing the first immunogenicmolecule or a moiety thereof;

[0404] 3) administering to the mammal a second immunogenic moleculecomprising an antibody, an antibody fragment or another targeting moietywhich binds to the target cells and a member of a ligand/anti-ligandpair, and having at least one moiety in common with the firstimmunogenic molecule;

[0405] 4) administering the sensitized effector cells produced in step 2to the mammal; and

[0406] 5) administering an effector cell growth factor bound to a memberof the ligand/anti-ligand pair that is complementary to the memberadministered in step 2.

[0407] In this aspect of the present invention, the targeting moietyserves as a carrier to implant a strong predetermined antigenic signalon the target cells, ensuring that those target cells are sufficiently“immunogenic” for subsequent therapy. The engineered antigen bound tothe target cells in vivo attracts subsequently administeredpresensitized effector cells to the target cells. In effect, thisprocedure constitutes an amplification of the tumor-associated antigen.The ligand/anti-ligand pair conjugate components are employed both todeliver effector cell growth factor to the target site and to avoidtoxicity associated with circulating effector cell growth factor bydecoupling the slow targeting moiety localization step from delivery ofthe growth factor.

[0408] An alternative method for eradicating target cells within amammal comprises administering to the mammal an immunogenic moleculecomprising an antibody- or other targeting moiety-ligand/anti-ligandpair member-immunogen conjugate which binds to the target cells in vivo.Subsequently, the complementary member of the ligand/anti-ligand pair isadministered while conjugated to an effector cell growth factor. This invivo embodiment may be employed when the immunogenic molecule is capableof inducing a therapeutic response in vivo. In this case, the antigenicsignal is not predetermined, but is sufficiently strong to recruit atherapeutically effective number of effector cells to the target site.The binding of the immunogenic molecule to the target cells within themammal induces a cytotoxic effector cell response directed at the targetcells. Delivery of effector cell growth factor to the target siteenhances the activity of the effector cells at that site.

[0409] In accordance with the present invention, effector cells areextracted from a mammal or recruited from within a mammal. Theseeffector cells are cells capable of being “sensitized” such that thesensitized effector cells recognize a particular antigen, and have acytotoxic effect on target cells bearing that antigen. The effectorcells useful in the practice of this aspect of the present invention arecells designated as “committed T-cells,” along with a variety ofcytotoxic cells designated otherwise, including LAK cells, naturalkiller (NK) cells and other leukocytes and macrophages which couldpotentially be sensitized and manipulated similarly. In a preferredembodiment of the present invention, the effector cells are cytotoxicT-lymphocytes, also called cytotoxic T-cells.

[0410] Effector cells can be extracted from the mammal to be treated byany suitable procedure for collecting those cells from the patient andseparating the desired effector cells from other cell types orbiological materials which may be simultaneously extracted. Conventionalcell harvesting or cytopheresis (e.g., leukopheresis) procedures may beused, and the effector cell extraction procedure may be repeated toobtain a larger quantity of effector cells. For example, the mammal maybe subjected to one leukapheresis procedure per day for one or more days(e.g., for five days to collect a large number of effector cells such asT-cells).

[0411] The effector cell growth factor used may vary according to thetype of effector cells employed, and may be any growth factor whichstimulates the propagation of the cultured cells or enhances theactivation thereof so that sensitized cytotoxic effector cells areproduced. Among suitable growth factors are monokines for monocytes andlymphokines (e.g., IL-2) for lymphocytes such as T-lymphocytes).

[0412] The first immunogenic molecule (that with which the effectorcells are incubated) may be the same as or different from the secondimmunogenic molecule (that which is administered to the mammal),provided that the two immunogenic molecules have at least one moiety incommon. The proviso ensures that effector cells sensitized in vitro inthe presence of the first immunogenic molecule will recognize the secondimmunogenic molecule in vivo.

[0413] When the targeting moiety, the ligand/anti-ligand pair member orthe combination thereof is sufficiently immunogenic (i.e., is capable ofinducing sensitization of effector cells), the targetingmoiety-ligand/anti-ligand pair member conjugate may serve as the firstas well as the second immunogenic molecule. Alternatively, asufficiently immunogenic portion of the conjugate may be used as thefirst immunogenic molecule with the entire conjugate serving as thesecond immunogenic molecule.

[0414] When the targeting moiety-ligand/anti-ligand pair memberconjugate or any portion thereof is not sufficiently immunogenic (i.e.,is incapable of inducing sensitization of effect of cells) variouscompounds may be attached to the targeting moiety or ligand/anti-ligandpair member to increase the immunogenicity thereof. In this case, theimmunogen-targeting moiety-ligand/anti-ligand pair member conjugate isadministered as the second immunogenic molecule.

[0415] Among the types of compounds that may be attached to a targetingmoiety or ligand/anti-ligand pair member to increase the immunogenicitythereof are haptens or polypeptides which are “foreign” to the intendedmammalian recipient (such as antigens, mitogens, other foreign proteinsor fragments thereof, peptides that activate cytotoxic T-cells or thelike). In some circumstances, attachment of the immunogen to thetargeting moiety or ligand/anti-ligand pair member through a spacer(i.e., a linker molecule that serves to physically separate thecomponents) may increase the immunogenicity of the linked firstimmunogenic molecule.

[0416] Haptens that are useful in the practice of the present inventioninclude benzoate groups, nitrophenol groups, other small molecules suchas acetic acid and derivatives thereof, penicillinic acid andderivatives thereof, sulfanilic acid derivatives, hexoseamines,ribonucleotides, ribonucleosides, isocyanates, isothiocyanates and thelike. Preferred haptens are benzoic acid, dinitro-chlorobenzene,dinitro-fluorobenzene, picrylchloride, p-aminobenzenearsonate,p-azo-benzenearsonate, acetic acid, dinitrophenol, trinitrophenol,trinitrophenol-epsilon-aminocaproic acid,3-iodo-4-hydroxyl-5-nitrophenylacetic acid, p-hydroxyphenylacetic acid,p-sulfanilic acid, 2,4-diisocyanate, fluorescein isothiocyanate,N-acetyl-glucosamine and the like.

[0417] “Foreign” polypeptides useful in the practice of this aspect ofthe present invention include flagellins, fragments of keyhole limpethemocyanin, histocompatibility antigens, bacterial cell surfaceproteins, viral coat proteins, fragments thereof and the like. Smallpolypeptide fragments are generally Preferred in order to avoidinterference with the biodistribution of the targeting moiety-containingconjugate. T-cell mitogens such as the proteins pokeweed antiviralprotein and phytohemagglutinin also may be used.

[0418] A specific example of the in vitro sensitization embodiment ofthis aspect of the present invention may be described as follows:

[0419] 1) extracting effector cells (or precursors thereof) from themammal;

[0420] 2) cultivating the extracted effector cells in vitro withinterleukin-2 (IL-2) and avidin or streptavidin, thereby producingsensitized effector cells recognizing avidin or streptavidin or a moietythereof.;

[0421] 3) administering to the mammal a targeting moiety-avidin or-streptavidin conjugate;

[0422] 4) administering the sensitized effector cells produced in step 2to the mammal; and

[0423] 5) administering an IL-2-biotin conjugate. A three-steppretargeting approach may be designed for the practice of this aspect ofthe present invention.

[0424] Another specific example of an in vitro embodiment of the presentinvention involves the following steps:

[0425] 1) extracting effector cells (or precursors thereof) from amammal;

[0426] 2) cultivating the effector cells with IL-2;

[0427]3) conjugating the cultivated effector cells with biotin;

[0428] 4) administering a targeting moiety-avidin or targetingmoiety-streptavidin to the mammal, wherein the targeting moiety isspecific for the cells upon which effector cell action is desired; and

[0429] 5) administering effector cell-biotin conjugate to the mammal.

[0430] In this embodiment of the present invention, steps 3 and 4 may beconducted in reverse order. Also, a clearing agent may be administeredbetween steps 4 and 5. In addition, a three-step pretargeting protocolmay be employed.

[0431] A specific example of the in vivo sensitization embodiment ofthis aspect of the present invention may be described as follows:

[0432] 1) administering to the mammal a hapten-targeting moiety-avidinor -streptavidin conjugate to induce sensitization of effector cells;and

[0433] 2) administering to the mammal an IL-2-biotin conjugate.

[0434] A three-step pretargeting protocol may also be designed for thepractice of this embodiment of the present invention.

[0435] Another protocol employing the concepts of the effector cellsensitization involves principles incident to organ transplantation. TheHLA system in humans, including class I and class II antigens,corresponds to the MHC complex of mice, for example, and is a majorfactor in self/non-self recognition. For this reason, HLA matching isconducted in selecting candidates for organ transplants. Persons withmore similar HLA (i.e., twins, siblings, parents and the like) are themost preferably organ transplant donors, because the donated organ isless likely to be recognized as non-self by the recipient.

[0436] Some HLA antigens are relatively rare in humans, i.e., areinfrequently present in the human HLA display. For example, the HLA-B7antigen is present on only 5% of the human population. Consequently,HLA-B7 is recognized as non-self in 95% of humans. Such relatively rareantigens can therefore be targeted to target sites within an individualthat does not exhibit the antigen. Due to the presence of the antigen atthe target site, the target will be recognized as non-self by therecipient, thereby recruiting the recipient's immune system to combatthe target. Since some targets (e.g., certain tumors) are poorlyrecognized as non-self, the presence of a recognized non-self antigen atthat site will improve the recipient's immune system response to thetarget.

[0437] HLA antigen that is not present in a recipient can be deliveredto a target site by conjugation with a targeting moiety, as part of afusion protein with a targeting moiety or as part of a conjugate orfusion protein with a ligand or an anti-ligand in the two-step orthree-step pretargeting protocols described herein.

[0438] The invention is further described through presentation of thefollowing examples. These examples are offered by way of illustration,and not by way of limitation.

EXAMPLE I Synthesis of a Chelate-Biotin Conjugate

[0439] A chelating compound that contains an N₃S chelating core wasattached via an amide linkage to biotin. Radiometal labeling of anexemplary chelate-biotin conjugate is illustrated below.

[0440] The spacer group “X” permits the biotin portion of the conjugateto be sterically available for avidin binding. When “R¹” is a carboxylicacid substituent (for instance, CH₂COOH), the conjugate exhibitsimproved water solubility, and further directs in vivo excretion of theradiolabeled biotin conjugate toward renal rather than hepatobiliaryclearance.

[0441] Briefly, N-α-Cbz-N-Σ-t-BOC protected lysine was converted to thesuccinimidyl ester with NHS and DCC, and then condensed with asparticacid β-t-butyl ester. The resultant dipeptide was activated with NHS andDCC, and then condensed with glycine t-butyl ester. The Cbz group wasremoved by hydrogenolysis, and the amine was acylated usingtetrahydropyranyl mercaptoacetic acid succinimidyl ester, yieldingS-(tetrahydropyranyl)-mercaptoacetyl-lysine. Trifluoroacetic acidcleavage of the N-t-BOC group and t-butyl esters, followed bycondensation with LC-biotin-NHS ester provided (Σ-caproylamidebiotin)-aspartyl glycine. This synthetic method is illustrated below.

[0442]¹H NMR: (CD₃OD, 200 MHz Varian): 1.25-1.95 (m, 24H), 2.15-2.25(broad t, 4H), 2.65-3.05 (m, 4H), 3.30-3.45 (dd, 2H), 3.50-3.65 (ddd,2H), 3.95 (broad s, 2H), 4.00-4.15 (m, 1H), 4.25-4.35 (m, 1H), 4.45-4.55(m, 1H), 4.7-5.05 (m overlapping with HOD).

[0443] Elemental Analysis: C, H, N for C₃₅H₅₇N₇O₁₁S₂.H₂O calculated:50.41, 7.13, 11.76 found: 50.13, 7.14, 11.40

EXAMPLE II Preparation of a Technetium or Rhenium RadiolabeledChelate-Biotin Conjugate

[0444] The chelate-biotin conjugate of Example I was radiolabeled witheither ^(99m)Tc pertechnetate or ¹⁸⁶Re perrhenate. Briefly, ^(99m)Tcpertechnetate was reduced with stannous chloride in the presence ofsodium gluconate to form an intermediate Tc-gluconate complex. Thechelate-biotin conjugate of Example I was added and heated to 100° C.for 10 min at a pH of about 1.8 to about 3.3. The solution wasneutralized to a pH of about 6 to about 8, and yielded anN₃S-coordinated ^(99m)Tc-chelate-biotin conjugate. C-18 HPLC gradientelution using 5-60% acetonitrile in it acetic acid demonstrated twoanomers at 97% or greater radiochemical yield using δ (gamma ray)detection.

[0445] Alternatively, ¹⁸⁶Re perrhenate was spiked with cold ammoniumperrhenate, reduced with stannous chloride, and complexed with citrate.The chelate-biotin conjugate of Example I was added and heated to 90° C.for 30 min at a pH of about 2 to 3. The solution was neutralized to a pHof about 6 to about 8, and yielded an N₃S-coordinated¹⁸⁶Re-chelate-biotin conjugate. C-18 HPLC gradient elution using 5-60%acetonitrile in 1% acetic acid resulted in radiochemical yields of85-90%. Subsequent purification over a C-18 reverse phase hydrophobiccolumn yielded material of 99% purity.

EXAMPLE III In Vitro Analysis of Radiolabeled Chelate-Biotin Conjugates

[0446] Both the ^(99m)Tc- and ¹⁸⁶Re-chelate-biotin conjugates wereevaluated in vitro. When combined with excess avidin (about 100-foldmolar excess), 100% of both radiolabeled biotin conjugates complexedwith avidin.

[0447] A ^(99m)Tc-biotin conjugate was subjected to various chemicalchallenge conditions. Briefly, ^(99m)Tc-chelate-biotin conjugates werecombined with avidin and passed over a 5 cm size exclusion gelfiltration column. The radiolabeled biotin-avidin complexes weresubjected to various chemical challenges (see Table 1), and theincubation mixtures were centrifuged through a size exclusion filter.The percent of radioactivity retained (indicatingavidin-biotin-associated radiolabel) is presented in Table 1. Thus, uponchemical challenge, the radiometal remained associated with themacromolecular complex. TABLE 1 Chemical Challenge of ^(99m)Tc-Chelate-Biotin-Avidin Complexes Challenge % Radioactivity Retained Medium pH 1h, 37° C. 18 h, RT PBS 7.2 99 99 Phosphate 8.0 97 97 10 mM cysteine 8.092 95 10 mM DTPA 8.0 99 98 0.2 M carbonate 10.0 97 94

[0448] In addition, each radiolabeled biotin conjugate was incubated atabout 50 μg/ml with serum; upon completion of the incubation, thesamples were subjected to instant thin layer chromatography (ITLC) in80% methanol. Only 2-4% of the radioactivity remained at the origin(i.e., associated with protein); this percentage was unaffected by theaddition of exogenous biotin. When the samples were analyzed using sizeexclusion H-12 FPLC with 0.2 M phosphate as mobile phase, no associationof radioactivity with serum macromolecules was observed.

[0449] Each radiolabeled biotin conjugate was further examined using acompetitive biotin binding assay. Briefly, solutions containing varyingratios of D-biotin to radiolabeled biotin conjugate were combined withlimiting avidin at a constant total biotin:avidin ratio. Avidin bindingof each radiolabeled biotin conjugate was determined by ITLC, and wascompared to the theoretical maximum stoichiometric binding (asdetermined by the HABA spectrophotometric assay of Green, Biochem. J.94:23c-24c, 1965). No significant difference in avidin binding wasobserved between each radiolabeled biotin conjugate and D-biotin.

EXAMPLE IV In vivo Analysis of Radiolabeled Chelate-Biotin ConjugatesAdministered After Antibody Pretargeting

[0450] The ¹⁸⁶Re-chelate-biotin conjugate of Example I was studied in ananimal model of a three-step antibody pretargeting protocol. Generally,this protocol involved: (i) prelocalization of biotinylated monoclonalantibody; (ii) administration of avidin for formation of a “sandwich” atthe target site and for clearance of residual circulating biotinylatedantibody; and (iii) administration of the 186Re-biotin conjugate fortarget site localization and rapid blood clearance.

[0451] A. Preparation and Characterization of Biotinylated Antibody

[0452] Biotinylated NR-LU-10 was prepared according to either of thefollowing procedures. The first procedure involved derivitization ofantibody via lysine E-amino groups. NR-LU-10 was radioiodinated attyrosines using chloramine T and either ¹²⁵I or ¹³¹I sodium iodide. Theradioiodinated antibody (5-10 mg/ml) was then biotinylated usingbiotinamido caproate NHS ester in carbonate buffer, pH 8.5, containing5% DMSO, according to the scheme below.

[0453] The impact of lysine biotinylation on antibody immunoreactivitywas examined. As the molar offering of biotin:antibody increased from5:1 to 40:1, biotin incorporation increased as expected (measured usingthe HABA assay and pronase-digested product) (Table 2, below). Percentof biotinylated antibody immunoreactivity as compared to native antibodywas assessed in a limiting antigen ELISA assay. The immunoreactivitypercentage dropped below 70% at a measured derivitization of 11.1:1;however, at this level of derivitization, no decrease was observed inantigen-positive cell binding (performed with LS-180 tumor cells atantigen excess). Subsequent experiments used antibody derivitized at abiotin:antibody ratio of 10:1. TABLE 2 Effect of Lysine Biotinylation onImmunoreactivity Molar Measured Offering Derivitization Immunoassessment(%) (Biotins/Ab) (Biotins/Ab) ELISA Cell Binding  5:1 3.4 86 10:1 8.5 73100 13:1 11.1 69 102 20:1 13.4 36 106 40:1 23.1 27

[0454] Alternatively, NR-LU-10 was biotinylated using thiol groupsgenerated by reduction of cystines. Derivitization of thiol groups washypothesized to be less compromising to antibody immunoreactivity.NR-LU-10 was radioiodinated using p-aryltin phenylate NHS ester(PIP-NHS) and either ¹²⁵I or ¹³¹I sodium iodide. Radioiodinated NR-LU-10was incubated with 25 mM dithiothreitol and purified using sizeexclusion chromatography. The reduced antibody (containing free thiolgroups) was then reacted with a 10- to 100-fold molar excess ofN-iodoacetyl-n′-biotinyl hexylene diamine in phosphate-buffered saline(PBS), pH 7.5, containing 5% DMSO (v/v). TABLE 3 Effect of ThiolBiotinylation on Immunoreactivity Molar Measured Offering DerivitizationImmunoassessment (%) (Biotins/Ab) (Biotins/Ab) ELISA Cell Binding 10:14.7 114 50:1 6.5 102 100 100:1  6.1 95 100

[0455] As shown in Table 3, at a 50:1 or greater biotin:antibody molaroffering, only 6 biotins per antibody were incorporated. No significantimpact on immunoreactivity was observed.

[0456] The lysine- and thiol-derivitized biotinylated antibodies(“antibody (lysine)” and “antibody (thiol)”, respectively) werecompared. Molecular sizing on size exclusion FPLC demonstrated that bothbiotinylation protocols yielded monomolecular (monomeric) IgGs.Biotinylated antibody (lysine) had an apparent molecular weight of 160kD, while biotinylated antibody (thiol) had an apparent molecular weightof 180 kD. Reduction of endogenous sulfhydryls (i.e., disulfides) tothiol groups, followed by conjugation with biotin, may produce asomewhat unfolded macromolecule. If so, the antibody (thiol) may displaya larger hydrodynamic radius and exhibit an apparent increase inmolecular weight by chromatographic analysis. Both biotinylated antibodyspecies exhibited 98% specific binding to immobilized avidin-agarose.

[0457] Further comparison of the biotinylated antibody species wasperformed using non-reducing SDS-PAGE, using a 4% stacking gel and a 5%resolving gel. Biotinylated samples were either radiolabeled orunlabeled and were combined with either radiolabeled or unlabeled avidinor streptavidin. Samples were not boiled prior to SDS-PAGE analysis. Thenative antibody and biotinylated antibody (lysine) showed similarmigrations; the biotinylated antibody (thiol) produced two species inthe 50-75 kD range. These species may represent two thiol-cappedspecies. Under these SDS-PAGE conditions, radiolabeled streptavidinmigrates as a 60 kD tetramer. When 400 μg/ml radiolabeled streptavidinwas combined with 50 μg/ml biotinylated antibody (analogous to“sandwiching” conditions in vivo), both antibody species formed largemolecular weight complexes. However, only the biotinylated antibody(thiol)-streptavidin complex moved from the stacking gel into theresolving gel, indicating a decreased molecular weight as compared tothe biotinylated antibody (lysine)-streptavidin complex.

[0458] B. Blood Clearance of Biotinylated Antibody Species

[0459] Radioiodinated biotinylated NR-LU-10 (lysine or thiol) wasintravenously administered to non-tumored nude mice at a dose of 100 μg.At 24 h post-administration of radioiodinated biotinylated NR-LU-10,mice were intravenously injected with either saline or 400 μg of avidin.With saline administration, blood clearances for both biotinylatedantibody species were biphasic and similar to the clearance of nativeNR-LU-10 antibody.

[0460] In the animals that received avidin intravenously at 24 h, thebiotinylated antibody (lysine) was cleared (to a level of 5% of injecteddose) within 15 min of avidin administration (avidin:biotin=10:1). Withthe biotinylated antibody (thiol), avidin administration (10:1 or 25:1)reduced the circulating antibody level to about 35% of injected doseafter two hours. Residual radiolabeled antibody activity in thecirculation after avidin administration was examined in vitro usingimmobilized biotin. This analysis revealed that 85% of the biotinylatedantibody was complexed with avidin. These data suggest that thebiotinylated antibody (thiol)-avidin complexes that were formed wereinsufficiently crosslinked to be cleared by the RES.

[0461] Blood clearance and biodistribution studies of biotinylatedantibody (lysine) 2 h post-avidin or post-saline administration wereperformed. Avidin administration significantly reduced the level ofbiotinylated antibody in the blood (see FIG. 1), and increased the levelof biotinylated antibody in the liver and spleen. Kidney levels ofbiotinylated antibody were similar.

EXAMPLE V In vivo Characterization of ¹⁸⁶Re-Chelate-Biotin Conjugates ina Three-Step Pretargeting Protocol

[0462] A ¹⁸⁶Re-chelate-biotin conjugate of Example I (MW≈1000; specificactivity=1-2 mCi/mg) was examined in a three-step pretargeting protocolin an animal model. More specifically, 18-22 g female nude mice wereimplanted subcutaneously with LS-180 human colon tumor xenografts,yielding 100-200 mg tumors within 10 days of implantation.

[0463] NR-LU-10 antibody (MW≈150 kD) was radiolabeled with¹²⁵I/Chloramine T and biotinylated via lysine residues (as described inExample IV.A, above). Avidin (MW≈66 kD) was radiolabeled with¹³¹I/PIP-NHS (as described for radioiodination of NR-LU-10 in ExampleIV.A., above). The experimental protocol was as follows: Group 1: Time0, inject 100 μg ¹²⁵I-labeled, biotinylated NR-LU-10 Time 24 h, inject400 μg ¹³¹I-labeled avidin Time 26 h, inject 60 μg ¹⁸⁶Re-chelate- biotinconjugate Group 2: Time 0, inject 400 μg ¹³¹I-labeled avidin (control)Time 2 h, inject 60 μg ¹⁸⁶Re-chelate- biotin conjugate Group 3: Time 0,inject 60 μg ¹⁸⁶Re-chelate- (control) biotin conjugate

[0464] The three radiolabels employed in this protocol are capable ofdetection in the presence of each other. It is also noteworthy that thesizes of the three elements involved are logarithmicallydifferent—antibody≅150,000; avidin≅66,000; and biotin≅1,000.Biodistribution analyses were performed at 2, 6, 24, 72 and 120 h afteradministration of the ¹⁸⁶Re-chelate-biotin conjugate.

[0465] Certain preliminary studies were performed in the animal modelprior to analyzing the ¹⁸⁶Re-chelate-biotin conjugate in a three-steppretargeting protocol. First, the effect of biotinylated antibody onblood clearance of avidin was examined. These experiments showed thatthe rate and extent of avidin clearance was similar in the presence orabsence of biotinylated antibody. Second, the effect of biotinylatedantibody and avidin on blood clearance of the ¹⁸⁶Re-chelate-biotinconjugate was examined; blood clearance was similar in the presence orabsence of biotinylated antibody and avidin. Further, antibodyimmunoreactivity was found to be uncompromised by biotinylation at thelevel tested.

[0466] Third, tumor uptake of biotinylated antibody administered at time0 or of avidin administered at time 24 h was examined. The results ofthis experimentation are shown in FIG. 1. At 25 h, about 350 pmol/gbiotinylated antibody was present at the tumor; at 32 h the level wasabout 300 pmol/g; at 48 h, about 200 pmol/g; and at 120 h, about 100pmol/g. Avidin uptake at the same time points was about 250, 150, 50 and0 pmol/g, respectively. From the same experiment, tumor to blood ratioswere determined for biotinylated antibody and for avidin. From 32 h to120 h, the ratios of tumor to blood were very similar.

[0467] Rapid and efficient removal of biotinylated antibody from theblood by complexation with avidin was observed. Within two hours ofavidin administration, a 10-fold reduction in blood pool antibodyconcentration was noted (FIG. 1), resulting in a sharp increase in tumorto blood ratios. Avidin is cleared rapidly, with greater than 90% of theinjected dose cleared from the blood within 1 hour after administration.The Re-186-biotin chelate is also very rapidly cleared, with greaterthan 99% of the injected dose cleared from the blood by 1 hour afteradministration.

[0468] The three-step pretargeting protocol (described for Group 1,above) was then examined. More specifically, tumor uptake of the¹⁸⁶Re-chelate-biotin conjugate in the presence or absence ofbiotinylated antibody and avidin was determined. In the absence ofbiotinylated antibody and avidin, the ¹⁸⁶Re-chelate-biotin conjugatedisplayed a slight peak 2 h post-injection, which was substantiallycleared from the tumor by about 5 h. In contrast, at 2 h post-injectionin the presence of biotinylated antibody and avidin (specific), the¹⁸⁶Re-chelate-biotin conjugate reached a peak in tumor approximately 7times greater than that observed in the absence of biotinylated antibodyand avidin. Further, the specifically bound ¹⁸⁶Re-chelate-biotinconjugate was retained at the tumor at significant levels for more than50 h. Tumor to blood ratios determined in the same experiment increasedsignificantly over time (i.e., T:B=≈8 at 30 h; ≈15 at 100 h; ≈35 at 140h).

[0469] Tumor uptake of the ¹⁸⁶Re-chelate-biotin conjugate has furtherbeen shown to be dependent on the dose of biotinylated antibodyadministered. At 0 μg of biotinylated antibody, about 200 pmol/g of¹⁸⁶Re-chelate-biotin conjugate was present at the tumor at 2 h afteradministration; at 50 μg antibody, about 500 pmol/g of¹⁸⁶Re-chelate-biotin conjugate; and at 100 μg antibody, about 1,300pmol/g of ¹⁸⁶Re-chelate-biotin conjugate.

[0470] Rhenium tumor uptake via the three-step pretargeting protocol wascompared to tumor uptake of the same antibody radiolabeled throughchelate covalently attached to the antibody (conventional procedure).The results of this comparison are depicted in FIG. 2. Blood clearanceand tumor uptake were compared for the chelate directly labeled rheniumantibody conjugate and for the three-step pretargeted sandwich. Areasunder the curves (AUC) and the ratio of AUC_(tumor)/AUC_(blood) weredetermined. For the chelate directly labeled rhenium antibody conjugate,the ratio of AUC_(tumor)/AUC_(blood)=24055/10235 or 2.35; for thethree-step pretargeted sandwich, the ratio ofAUC_(tumor)/AUC_(blood)=46764/6555 or 7.13.

[0471] Tumor uptake results are best taken in context with radioactivityexposure to the blood compartment, which directly correlates with bonemarrow exposure. Despite the fact that 100-fold more rhenium wasadministered to animals in the three-step protocol, the very rapidclearance of the small molecule (Re-186-biotin) from the blood minimizesthe exposure to Re-186 given in this manner. In the same matchedantibody dose format, direct labeled (conventional procedure) NR-LU-10whole antibody yielded greater exposure to rhenium than did the 100-foldhigher dose given in the three-step protocol. A clear increase in thetargeting ratio (tumor exposure to radioactivity:blood exposure toradioactivity—AUC_(tumor):AUC_(blood)) was observed for three-steppretargeting (approximately 7:1) in comparison to the direct labeledantibody approach (approximately 2.4:1).

EXAMPLE VI Preparation of Chelate-Biotin Conjugates Having ImprovedBiodistribution Properties

[0472] The biodistribution of ¹¹¹in-labeled-biotin derivatives variesgreatly with structural changes in the chelate and the conjugatinggroup. Similar structural changes may affect the biodistribution oftechnetium- and rhenium-biotin conjugates. Accordingly, methods forpreparing technetium- and rhenium-biotin conjugates having optimalclearance from normal tissue are advantageous.

[0473] A. Neutral MAMA Chelate/Conjugate

[0474] A neutral MAMA chelate-biotin conjugate is prepared according tothe following scheme.

[0475] The resultant chelate-biotin conjugate shows superior kidneyexcretion. Although the net overall charge of the conjugate is neutral,the polycarboxylate nature of the molecule generates regions ofhydrophilicity and hydrophobicity. By altering the number and nature ofthe carboxylate groups within the conjugate, excretion may be shiftedfrom kidney to gastrointestinal routes. For instance, neutral compoundsare generally cleared by the kidneys; anionic compounds are generallycleared through the GI system.

[0476] B. Polylysine Derivitization

[0477] Conjugates containing polylysine may also exhibit beneficialbiodistribution properties. With whole antibodies, derivitization withpolylysine may skew the biodistribution of conjugate toward liveruptake. In contrast, derivitization of Fab fragments with polylysineresults in lower levels of both liver and kidney uptake; blood clearanceof these conjugates is similar to that of Fab covalently linked tochelate. An exemplary polylysine derivitized chelate-biotin conjugate isillustrated below.

[0478] Inclusion of polylysine in radiometal-chelate-biotin conjugatesis therefore useful for minimizing or eliminating RES sequestrationwhile maintaining good liver and kidney clearance of the conjugate. Forimproved renal excretion properties, polylysine derivatives arepreferably succinylated following biotinylation. Polylysine derivativesoffer the further advantages of: (1) increasing the specific activity ofthe radiometal-chelate-biotin conjugate; (2) permitting control of rateand route of blood clearance by varying the molecular weight of thepolylysine polymer; and (3) increasing the circulation half-life of theconjugate for optimal tumor interaction.

[0479] Polylysine derivitization is accomplished by standardmethodologies. Briefly, poly-L-lysine is acylated according to standardamino group acylation procedures (aqueous bicarbonate buffer, pH 8,added biotin-NHS ester, followed by chelate NHS ester). Alternativemethodology involves anhydrous conditions using nitrophenyl esters inDMSO and triethyl amine. The resultant conjugates are characterized byUV and NMR spectra.

[0480] The number of biotins attached to polylysine is determined by theHABA assay. Spectrophotometric titration is used to assess the extent ofamino group derivitization. The radiometal-chelate-biotin conjugate ischaracterized by size exclusion.

[0481] C. Cleavable Linkage

[0482] Through insertion of a cleavable linker between the chelate andbiotin portion of a radiometal-chelate-biotin conjugate, retention ofthe conjugate at the tumor relative to normal tissue may be enhanced.More specifically, linkers that are cleaved by enzymes present in normaltissue but deficient or absent in tumor tissue can increase tumorretention. As an example, the kidney has high levels of γ-glutamyltransferase; other normal tissues exhibit in vivo cleavage of γ-glutamylprodrugs. In contrast, tumors are generally deficient in enzymepeptidases. The glutamyl-linked biotin conjugate depicted below iscleaved in normal tissue and retained in the tumor.

[0483] D. Serine Linker with O-Polar Substituent

[0484] Sugar substitution of N₃S chelates renders such chelates watersoluble. Sulfonates, which are fully ionized at physiological pH,improve water solubility of the chelate-biotin conjugate depicted below.

[0485] This compound is synthesized according to the standard reactionprocedures. Briefly, biocytin is condensed with N-t-BOC-(O-sulfonate orO-glucose) serine NHS ester to give N-t-BOC-(O-sulfonate or O-glucose)serine biocytinamide. Subsequent cleavage of the N-t-BOC group with TFAand condensation with ligand NHS ester in DMF with triethylamineprovides ligand-amidoserine(O-sulfonate or O-glucose)biocytinamide.

EXAMPLE VII

[0486] Preparation and Characterization of PIP-Radioiodinated Biotin

[0487] Radioiodinated biotin derivatives prepared by exposure ofpoly-L-lysine to excess NHS-LC-biotin and then to Bolton-HunterN-hydroxysuccinimide esters in DMSO has been reported. Afterpurification, this product was radiolabeled by the iodogen method (see,for instance, Del Rosario et al., J. Nucl. Med. 32:5, 1991, 993(abstr.)). Because of the high molecular weight of the resultantradioiodinated biotin derivative, only limited characterization ofproduct (i.e., radio-HPLC and binding to immobilized streptavidin) waspossible.

[0488] Preparation of radioiodinated biotin according to the presentinvention provides certain advantages. First, the radioiodobiotinderivative is a low molecular weight compound that is amenable tocomplete chemical characterization. Second, the disclosed methods forpreparation involve a single step and eliminate the need for apurification step.

[0489] Briefly, iodobenzamide derivatives corresponding to biocytin(R═COOH) and biotinamidopentylamine (R═H) were prepared according to thefollowing scheme. In this scheme, “X” may be any radiohalogen, including¹²⁵I, ¹³¹I, ¹²³I, ²¹¹At and the like.

[0490] Preparation of 1 was generally according to Wilbur et al., J.Nucl. Med. 30:216-26, 1989, using a tributyltin intermediate. Watersoluble carbodiimide was used in the above-depicted reaction, since theNHS ester 1 formed intractable mixtures with DCU. The NHS ester was notcompatible with chromatography; it was insoluble in organic and aqueoussolvents and did not react with biocytin in DMF or in buffered aqueousacetonitrile. The reaction between 1 and biocytin or 5-(biotinamido)pentylamine was sensitive to base. When the reaction of 1 and biocytinor the pentylamine was performed in the presence of triethylamine in hotDMSO, formation of more than one biotinylated product resulted. Incontrast, the reaction was extremely clean and complete when asuspension of 1 and biocytin (4 mg/ml) or the pentylamine (4 mg/ml) washeated in DMSO at 117° C. for about 5 to about 10 min. The resultant¹²⁵I-biotin derivatives were obtained in 94% radiochemical yield.Optionally, the radioiodinated products may be purified using C-18 HPLCand a reverse phase hydrophobic column. Hereinafter, the resultantradioiodinated products 2 are referred to as PIP-biocytin (R═COOH) andPIP-pentylamine (R═H).

[0491] Both iodobiotin derivatives 2 exhibited ≧95% binding toimmobilized avidin. Incubation of the products 2 with mouse serumresulted in no loss of the ability of 2 to bind to immobilized avidin.Biodistribution studies of 2 in male BALB/c mice showed rapid clearancefrom the blood (similar to ¹⁸⁶Re-chelate-biotin conjugates describedabove). The radioiodobiotin 2 had decreased hepatobiliary excretion ascompared to the ¹⁸⁶Re-chelate-biotin conjugate; urinary excretion wasincreased as compared to the ¹⁸⁶Re-chelate-biotin conjugate. Analysis ofurinary metabolites of 2 indicated deiodination and cleavage of thebiotin amide bond; the metabolites showed no binding to immobilizedavidin. In contrast, metabolites of the ¹⁸⁶Re-chelate-biotin conjugateappear to be excreted in urine as intact biotin conjugates. Intestinaluptake of 2 is <50% that of the ¹⁸⁶Re-chelate-biotin conjugate. Thesebiodistribution properties of 2 provided enhanced whole body clearanceof radioisotope and indicate the advantageous use of 2 withinpretargeting protocols.

[0492]¹³¹I-PIP-biocytin was evaluated in a two-step pretargetingprocedure in tumor-bearing mice. Briefly, female nude mice were injectedsubcutaneously with LS-180 tumor cells; after 7 d, the mice displayed50-100 mg tumor xenografts. At t=0, the mice were injected with 200 μgof NR-LU-10-avidin conjugate labeled with ¹²⁵I using PIP-NHS (seeExample IV.A.). At t=36 h, the mice received 42 μg of ¹³¹I-PIP-biocytin.The data showed immediate, specific tumor localization, corresponding to≈1.5 ¹³¹I-PIP-biocytin molecules per avidin molecule.

[0493] The described radiohalogenated biotin compounds are amenable tothe same types of modifications described in Example VI above for¹⁸⁶Re-chelate-biotin conjugates. In particular, the followingPIP-polylysine-biotin molecule is made by trace labeling polylysine with¹²⁵I-PIP, followed by extensive biotinylation of the polylysine.

[0494] Assessment of ¹²⁵I binding to immobilized avidin ensures that allradioiodinated species also contain at least an equivalent of biotin.

EXAMPLE VIII Preparation of Biotinylated Antibody (Thiol) ThroughEndogenous Antibody Sulfhydryl Groups or Sulfhydryl-Generating Compounds

[0495] Certain antibodies have available for reaction endogenoussulfhydryl groups. If the antibody to be biotinylated containsendogenous sulfhydryl groups, such antibody is reacted withN-iodoacetyl-n′-biotinyl hexylene diamine (as described in ExampleIV.A., above). The availability of one or more endogenous sulfhydrylgroups obviates the need to expose the antibody to a reducing agent,such as DTT, which can have other detrimental effects on thebiotinylated antibody.

[0496] Alternatively, one or more sulfhydryl groups are attached to atargeting moiety through the use of chemical compounds or linkers thatcontain a terminal sulfhydryl group. An exemplary compound for thispurpose is iminothiolane. As with endogenous sulfhydryl groups(discussed above), the detrimental effects of reducing agents onantibody are thereby avoided.

EXAMPLE IX Two-Step Pretargeting Methodology that Does Not InduceInternalization

[0497] A NR-LU-13-avidin conjugate is prepared as follows. Initially,avidin is derivitized with N-succinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC). SMCC-derivedavidin is then incubated with NR-LU-13 in a 1:1 molar ratio at pH 8.5for 16 h. Unreacted NR-LU-13 and SMCC-derived avidin are removed fromthe mixture using preparative size exclusion HPLC. Two conjugates areobtained as products—the desired 1:1 NR-LU-13-avidin conjugate as themajor product; and an incompletely characterized component as the minorproduct.

[0498] A ^(99m)Tc-chelate-biotin conjugate is prepared as in Example II,above. The NR-LU-13-avidin conjugate is administered to a recipient andallowed to clear from the circulation. One of ordinary skill in the artof radioimmunoscintigraphy is readily able to determine the optimal timefor NR-LU-13-avidin conjugate tumor localization and clearance from thecirculation. At such time, the ^(99m)Tc-chelate-biotin conjugate isadministered to the recipient. Because the ^(99m)Tc-chelate-biotinconjugate has a molecular weight of ≈1,000, crosslinking ofNR-LU-13-avidin molecules on the surface of the tumor cells isdramatically reduced or eliminated. As a result, the ^(99m)Tc diagnosticagent is retained at the tumor cell surface for an extended period oftime. Accordingly, detection of the diagnostic agent by imagingtechniques is optimized; further, a lower dose of radioisotope providesan image comparable to that resulting from the typical three-steppretargeting protocol.

[0499] Optionally, clearance of NR-LU-13-avidin from the circulation maybe accelerated by plasmapheresis in combination with a biotin affinitycolumn. Through use of such column, circulating NR-LU-13-avidin will beretained extracorporeally, and the recipient's immune system exposure toa large, proteinaceous immunogen (i.e., avidin) is minimized.

[0500] Exemplary methodology for plasmapheresis/column purificationuseful in the practice of the present invention is discussed in thecontext of reducing radiolabeled antibody titer in imaging and intreating tumor target sites in U.S. Pat. No. 5,078,673. Briefly, for thepurposes of the present invention, an example of an extracorporealclearance methodology may include the following steps:

[0501] administering a ligand- or anti-ligand-targeting moiety conjugateto a recipient;

[0502] after a time sufficient for localization of the administeredconjugate to the target site, withdrawing blood from the recipient by,for example, plasmapheresis;

[0503] separating cellular element from said blood to produce a serumfraction and returning the cellular elements to the recipient; and

[0504] reducing the titer of the administered conjugate in the serumfraction to produce purified serum;

[0505] infusing the purified serum back into the recipient.

[0506] Clearance of NR-LU-13-avidin is also facilitated byadministration of a particulate-type clearing agent (e.g., a polymericparticle having a plurality of biotin molecules bound thereto). Such aparticulate clearing agent preferably constitutes a biodegradablepolymeric carrier having a plurality of biotin molecules bound thereto.Particulate clearing agents of the present invention exhibit thecapability of binding to circulating administered conjugate and removingthat conjugate from the recipient. Particulate clearing agents of thisaspect of the present invention may be of any configuration suitable forthis purpose. Preferred particulate clearing agents exhibit one or moreof the following characteristics:

[0507] microparticulate (e.g., from about 0.5 micrometers to about 100micrometers in diameter, with from about 0.5 to about 2 micrometers morepreferred), free flowing powder structure;

[0508] biodegradable structure designed to biodegrade over a period oftime between from about 3 to about 180 days, with from about 10 to about21 days more preferred, or non-biodegradable structure;

[0509] biocompatible with the recipients physiology over the course ofdistribution, metabolism and excretion of the clearing agent, morepreferably including biocompatible biodegradation products;

[0510] and capability to bind with one or more circulating conjugates tofacilitate the elimination or removal thereof from the recipient throughone or more binding moieties (preferably, the complementary member ofthe ligand/anti-ligand pair). The total molar binding capacity of theparticulate clearing agents depends upon the particle size selected andthe ligand or anti-ligand substitution ratio. The binding moieties arecapable of coupling to the surface structure of the particulate dosageform through covalent or non-covalent modalities as set forth herein toprovide accessible ligand or anti-ligand for binding to its previouslyadministered circulating binding pair member.

[0511] Preferable particulate clearing agents of the present inventionare biodegradable or non-biodegradable microparticulates. Morepreferably, the particulate clearing agents are formed of a polymercontaining matrix that biodegrades by random, nonenzymatic, hydrolyticscissioning.

[0512] Polymers derived from the condensation of alpha hydroxycarboxylicacids and related lactones are more preferred for use in the presentinvention. A particularly preferred moiety is formed of a mixture ofthermoplastic polyesters (e.g., polylactide or polyglycolide) or acopolymer of lactide and glycolide components, such aspoly(lactide-co-glycolide). An exemplary structure, a randompoly(DL-lactide-co-glycolide), is shown below, with the values of x andy being manipulable by a practitioner in the art to achieve desirablemicroparticulate properties.

[0513] Other agents suitable for forming particulate clearing agents ofthe present invention include polyorthoesters and polyacetals (PolymerLetters, 18:293, 1980) and polyorthocarbonates (U.S. Pat. No. 4,093,709)and the like.

[0514] Preferred lactic acid/glycolic acid polymer containing matrixparticulates of the present invention are prepared by emulsion-basedprocesses, that constitute modified solvent extraction processes such asthose described by Cowsar et al., “Poly(Lactide-Co-Glycolide)Microcapsules for Controlled Release of Steroids,” Methods Enzymology,112:101-116, 1985 (steroid entrapment in microparticulates); Eldridge etal., “Biodegradable and Biocompatible Poly(DL-Lactide-Co-Glycolide)Microspheres as an Adjuvant for Staphylococcal Enterotoxin B ToxoidWhich Enhances the Level of Toxin-Neutralizing Antibodies,” Infectionand Immunity, 59:2978-2986, 1991 (toxoid entrapment); Cohen et al.,“Controlled Delivery Systems for Proteins Based on Poly(Lactic/GlycolicAcid) Microspheres,” Pharmaceutical Research, 8(6):713-720, 1991 (enzymeentrapment); and Sanders et al., “Controlled Release of a LuteinizingHormone-Releasing Hormone Analogue from Poly(D,L-Lactide-Co-Glycolide)Microspheres,” J. Pharmaceutical Science, 73(9):1294-1297, 1984 (peptideentrapment).

[0515] In general, the procedure for forming particulate clearing agentsof the present invention involves dissolving the polymer in ahalogenated hydrocarbon solvent and adding an additional agent that actsas a solvent for the halogenated hydrocarbon solvent but not for thepolymer. The polymer precipitates out from the polymer-halogenatedhydrocarbon solution. Following particulate formation, they are washedand hardened with an organic solvent. Water washing and aqueousnon-ionic surfactant washing steps follow, prior to drying at roomtemperature under vacuum.

[0516] For biocompatibility purposes, particulate clearing agents aresterilized prior to packaging, storage or administration. Sterilizationmay be conducted in any convenient manner therefor. For example, theparticulates can be irradiated with gamma radiation, provided thatexposure to such radiation does not adversely impact the structure orfunction of the binding moiety attached thereto. If the binding moietyis so adversely impacted, the particulate clearing agents can beproduced under sterile conditions.

[0517] The preferred lactide/glycolide structure is biocompatible withthe mammalian physiological environment. Also, these preferred sustainedrelease dosage forms have the advantage that biodegradation thereofforms lactic acid and glycolic acid, both normal metabolic products ofmammals.

[0518] Functional groups required for binding moiety-particulatebonding, are optionally included in the particulate structure, alongwith the non-degradable or biodegradable polymeric units. Functionalgroups that are exploitable for this purpose include those that arereactive with ligands or anti-ligands, such as carboxyl groups, aminegroups, sulfhydryl groups and the like. Preferred binding enhancementmoieties include the terminal carboxyl groups of the preferred(lactide-glycolide) polymer containing matrix or the like. Apractitioner in the art is capable of selecting appropriate functionalgroups and monitoring conjugation reactions involving those functionalgroups.

[0519] Advantages garnered through the use of particulate clearingagents of the type described above are as follows:

[0520] particles in the “micron” size range localize in the RES andliver, with galactose derivatization or charge modification enhancementmethods for this capability available, and, preferably, are designed toremain in circulation for a time sufficient to perform the clearancefunction;

[0521] the size of the particulates facilitates central vascularcompartment retention thereof, substantially precluding equilibrationinto the peripheral or extravascular compartment;

[0522] desired substituents for ligand or anti-ligand binding to theparticulates can be introduced into the polymeric structure;

[0523] ligand- or anti-ligand-particulate linkages having desiredproperties (e.g., serum biotinidase resistance thereby reducing therelease of biotin metabolite from a particle-biotin clearing agent) and

[0524] multiple ligands or anti-ligands can be bound to the particles toachieve optimal cross-linking of circulating targeting agent-ligand or-anti-ligand conjugate and efficient clearance of cross-linked species.This advantage is best achieved when care is taken to preventparticulate aggregation both in storage and upon in vivo administration.

[0525] Clearance of NR-LU-13-avidin may also be accelerated by anarterially inserted proteinaceous or polymeric multiloop device. Acatheter-like device, consisting of thin loops of synthetic polymer orprotein fibers derivitized with biotin, is inserted into a major artery(e.g., femoral artery) to capture NR-LU-13-avidin. Since the total bloodvolume passes through a major artery every 70 seconds, the in situclearing device is effective to reduce circulating NR-LU-13-avidinwithin a short period of time. This device offers the advantages thatNR-LU-13-avidin is not processed through the RES; removal ofNR-LU-13-avidin is controllable and measurable; and fresh devices withundiminished binding capacity are insertable as necessary. Thismethodology is also useful with intraarterial administration embodimentsof the present invention.

[0526] An alternative procedure for clearing NR-LU-13-avidin from thecirculation without induction of internalization involves administrationof biotinylated, high molecular weight molecules, such as liposomes, IgMand other molecules that are size excluded from ready permeability totumor sites. When such biotinylated, high molecular weight moleculesaggregate with NR-LU-13-avidin, the aggregated complexes are readilycleared from the circulation via the RES.

EXAMPLE X Enhancement of Therapeutic Agent Internalization ThroughAvidin Crosslinking

[0527] The ability of multivalent avidin to crosslink two or more biotinmolecules (or chelate-biotin conjugates) is advantageously used toimprove delivery of therapeutic agents. More specifically, avidincrosslinking induces internalization of crosslinked complexes at thetarget cell surface.

[0528] Biotinylated NR-CO-04 (lysine) is prepared according to themethods described in Example IV.A., above. Doxorubicin-avidin conjugatesare prepared by standard conjugation chemistry. The biotinylatedNR-CO-04 is administered to a recipient and allowed to clear from thecirculation. One of ordinary skill in the art of radioimmunotherapy isreadily able to determine the optimal time for biotinylated NR-CO-04tumor localization and clearance from the circulation. At such time, thedoxorubicin-avidin conjugate is administered to the recipient. Theavidin portion of the doxorubicin-avidin conjugate crosslinks thebiotinylated NR-CO-04 on the cell surface, inducing internalization ofthe complex. Thus, doxorubicin is more efficiently delivered to thetarget cell.

[0529] In a first alternative protocol, a standard three-steppretargeting methodology is used to enhance intracellular delivery of adrug to a tumor target cell. By analogy to the description above,biotinylated NR-LU-05 is administered, followed by avidin (for bloodclearance and to form the middle layer of the sandwich at the targetcell-bound biotinylated antibody). Shortly thereafter, and prior tointernalization of the biotinylated NR-LU-05-avidin complex, amethotrexate-biotin conjugate is administered.

[0530] In a second alternative protocol, biotinylated NR-LU-05 isfurther covalently linked to methotrexate. Subsequent administration ofavidin induces internalization of the complex and enhances intracellulardelivery of drug to the tumor target cell.

[0531] In a third alternative protocol, NR-CO-04-avidin is administeredto a recipient and allowed to clear from the circulation and localize atthe target site. Thereafter, a polybiotinylated species (such asbiotinylated poly-L-lysine, as in Example IV.B., above) is administered.In this protocol, the drug to be delivered may be covalently attached toeither the antibody-avidin component or to the polybiotinylated species.The polybiotinylated species induces internalization of the(drug)-antibody-avidin-polybiotin-(drug) complex.

EXAMPLE XI Targeting Moiety-Anti-Ligand Conjugate for Two-StepPretargeting in vivo

[0532] A. Preparation of SMCC-Derivitized Streptavidin. 31 mg (0.48μmol) streptavidin was dissolved in 9.0 ml PBS to prepare a finalsolution at 3.5 mg/ml. The pH of the solution was adjusted to 8.5 byaddition of 0.9 ml of 0.5 M borate buffer, pH 8.5. A DMSO solution ofSMCC (3.5 mg/ml) was prepared, and 477 μl (4.8 μmol) of this solutionwas added dropwise to the vortexing protein solution. After 30 minutesof stirring, the solution was purified by G-25 (PD-10, Pharmacia,Piscataway, N.J.) column chromatography to remove unreacted orhydrolyzed SMCC. The purified SMCC-derivitized streptavidin was isolated(28 mg, 1.67 mg/ml).

[0533] B. Preparation of DTT-reduced NR-LU-10. To 77 mg NR-LU-10 (0.42μmol) in 15.0 ml PBS was added 1.5 ml of 0.5 M borate buffer, pH 8.5. ADTT solution, at 400 mg/ml (165 μl) was added to the protein solution.After stirring at room temperature for 30 minutes, the reduced antibodywas purified by G-25 size exclusion chromatography. Purified DTT-reducedNR-LU-l0 was obtained (74 mg, 2.17 mg/ml).

[0534] C. Conjugation of SMCC-streptavidin to DTT-reduced NR-LU-10.DTT-reduced NR-LU-10 (63 mg, 29 ml, 0.42 μmol) was diluted with 44.5 mlPBS. The solution of SMCC-streptavidin (28 mg, 17 ml, 0.42 μmol) wasadded rapidly to the stirring solution of NR-LU-10. Total proteinconcentration in the reaction mixture was 1.0 mg/ml. The progress of thereaction was monitored by HPLC (Zorbax® GF-250, available from MacMod).After approximately 45 minutes, the reaction was-quenched by addingsolid sodium tetrathionate to a final concentration of 5 mM.

[0535] D. Purification of conjugate. For small scale reactions,monosubstituted conjugate was obtained using HPLC Zorbax (preparative)size exclusion chromatography. The desired monosubstituted conjugateproduct eluted at 14.0-14.5 min (3.0 ml/min flow rate), while unreactedNR-LU-10 eluted at 14.5-15 min and unreacted derivitized streptavidineluted at 19-20 min.

[0536] For larger scale conjugation reactions, monosubstituted adduct isisolatable using DEAE ion exchange chromatography. After concentrationof the crude conjugate mixture, free streptavidin was removed therefromby eluting the column with 2.5% xylitol in sodium borate buffer, pH 8.6.The bound unreacted antibody and desired conjugate were thensequentially eluted from the column using an increasing salt gradient in20 mM diethanolamine adjusted to pH 8.6 with sodium hydroxide.

[0537] E. Characterization of Conjugate.

[0538] 1. HPLC size exclusion was conducted as described above withrespect to small scale purification.

[0539] 2. SDS-PAGE analysis was performed using 5% polyacrylamide gelsunder non-denaturing conditions. Conjugates to be evaluated were notboiled in sample buffer containing SDS to avoid dissociation ofstreptavidin into its 15 kD subunits. Two product bands were observed onthe gel, which correspond to the mono- and di-substituted conjugates.

[0540] 3. Immunoreactivity was assessed, for example, by competitivebinding ELISA as compared to free antibody. Values obtained were within10% of those for the free antibody.

[0541] 4. Biotin binding capacity was assessed, for example, bytitrating a known quantity of conjugate withp-[I-125]iodobenzoylbiocytin. Saturation of the biotin binding sites wasobserved upon addition of 4 equivalences of the labeled biocytin.

[0542] 5. In vivo studies are useful to characterize the reactionproduct, which studies include, for example, serum clearance profiles,ability of the conjugate to target antigen-positive tumors, tumorretention of the conjugate over time and the ability of a biotinylatedmolecule to bind streptavidin conjugate at the tumor. These datafacilitate determination that the synthesis resulted in the formation ofa 1:1 streptavidin-NR-LU-10 whole antibody conjugate that exhibits bloodclearance properties similar to native NR-LU-10 whole antibody, andtumor uptake and retention properties at least equal to native NR-LU-10.

[0543] For example, FIG. 3 depicts the tumor uptake profile of theNR-LU-10-streptavidin conjugate (LU-10-StrAv) in comparison to a controlprofile of native NR-LU-10 whole antibody. LU-10-StrAv was radiolabeledon the streptavidin component only, giving a clear indication thatLU-10-StrAv localizes to target cells as efficiently as NR-LU-10 wholeantibody itself.

EXAMPLE XII Two-Step Pretargeting in vivo

[0544] A ¹⁸⁶Re-chelate-biotin conjugate (Re-BT) of Example I (MW≈1000;specific activity=1-2 mCi/mg) and a biotin-iodine-131 small molecule,PIP-Biocytin (PIP-BT, MW approximately equal to 602; specificactivity=0.5-1.0 mCi/mg), as discussed in Example VII above, wereexamined in a three-step pretargeting protocol in an animal model, asdescribed in Example V above. Like Re-BT, PIP-BT has the ability to bindwell to avidin and is rapidly cleared from the blood, with a serumhalf-life of about 5 minutes. Equivalent results were observed for bothmolecules in the two-step pretargeting experiments described herein.

[0545] NR-LU-10 antibody (MW≈150 kD) was conjugated to streptavidin(MW≈66 kD) (as described in Example XI above) and radiolabeled with¹²⁵I/PIP-NHS (as described for radioiodination of NR-LU-10 in ExampleIV.A., above). The experimental protocol was as follows:

[0546] Time 0 inject (i.v.) 200 μg NR-LU-10-StrAv conjugate;

[0547] Time 24-48 h inject (i.v.) 60-70 fold molar excess ofradiolabeled biotinyl molecule ;

[0548] and perform biodistributions at 2, 6, 24, 72, 120 hours afterinjection of radiolabeled biotinyl molecule

[0549] NR-LU-10-streptavidin has shown very consistent patterns of bloodclearance and tumor uptake in the LS-180 animal model. A representativeprofile is shown in FIG. 4. When either PIP-BT or Re-BT is administeredafter allowing the LU-10-StrAv conjugate to localize to target cellsites for at least 24 hours, the tumor uptake of therapeuticradionuclide is high in both absolute amount and rapidity. For PIP-BTadministered at 37 hours following LU-10-StrAv (I-125) administration,tumor uptake was above 500 pMOL/G at the 40 hour time point and peakedat about 700 pMOL/G at 45 hours post-LU-10-StrAv administration.

[0550] This almost instantaneous uptake of a small molecule therapeuticinto tumor in stoichiometric amounts comparable to the antibodytargeting moiety facilitates utilization of the therapeutic radionuclideat its highest specific activity. Also, the rapid clearance ofradionuclide that is not bound to LU-10-StrAv conjugate permits anincreased targeting ratio (tumor:blood) by eliminating the slow tumoraccretion phase observed with directly labeled antibody conjugates. Thepattern of radionuclide tumor retention is that of whole antibody, whichis very persistent.

[0551] Experimentation using the two-step pretargeting approach andprogressively lower molar doses of radiolabeled biotinyl molecule wasalso conducted. Uptake values of about 20% ID/G were achieved atno-carrier added (high specific activity) doses of radiolabeled biotinylmolecules. At less than saturating doses, circulating LU-10-StrAv wasobserved to bind significant amounts of administered radiolabeledbiotinyl molecule in the blood compartment.

EXAMPLE XIII Asialoorosomucoid Clearing Agent and Two-Step Pretargeting

[0552] In order to maximize the targeting ratio (tumor:blood), clearingagents were sought that are capable of clearing the blood pool oftargeting moiety-anti-ligand conjugate (e.g., LU-10-StrAv), withoutcompromising the ligand binding capacity thereof at the target sites.One such agent, biotinylated asialoorosomucoid, which employs theavidin-biotin interaction to conjugate to circulating LU-10-StrAv, wastested.

[0553] A. Derivitization of orosomucoid. 10 mg human orosomucoid (SigmaN-9885) was dissolved in 3.5 ml of pH 5.5 0.1 M sodium acetate buffercontaining 160 mM NaCl. 70 μl of a 2% (w/v) CaCl solution in deionized(D.I.) water was added and 11 μl of neuraminidase (Sigma N-7885), 4.6U/ml, was added. The mixture was incubated at 37° C. for 2 hours, andthe entire sample was exchanged over a Centricon-10® ultrafiltrationdevice (available from Amicon, Danvers, Mass.) with 2 volumes of PBS.The asialoorosomucoid and orosomucoid starting material wereradiolabeled with I-125 using PIP technology, as described in Example IVabove.

[0554] The two radiolabeled preparations were injected i.v. into femaleBALB/c mice (20-25 g), and blood clearance was assessed by serialretro-orbital eye bleeding of each group of three mice at 5, 10, 15 and30 minutes, as well as at 1, 2 and 4 hours post-administration. Theresults of this experiment are shown in FIG. 5, with asialoorosomucoidclearing more rapidly than its orosomucoid counterpart.

[0555] In addition, two animals receiving each compound were sacrificedat 5 minutes post-administration and limited biodistributions wereperformed. These results are shown in FIG. 6. The most striking aspectsof these data are the differences in blood levels (78% for orosomucoidand 0.4% for asialoorosomucoid) and the specificity of uptake ofasialoorosomucoid in the liver (86%), as opposed to other tissues.

[0556] B. Biotinylation of asialoorosomucoid clearing agent andorosomucoid control. 100 μl of 0.2 M sodium carbonate buffer, pH 9.2,was added to 2 mg (in 1.00 ml PBS) of PIP-125-labeled orosomucoid and to2 mg PIP-125-labeled asialoorosomucoid. 60 μl of a 1.85 mg/ml solutionof NHS-amino caproate biotin in DMSO was then added to each compound.The reaction mixtures were vortexed and allowed to sit at roomtemperature for 45 minutes. The material was purified by size exclusioncolumn chromatography (PD-10, Pharmacia) and eluted with PBS. 1.2 mlfractions were taken, with fractions 4 and 5 containing the majority ofthe applied radioactivity (>95%). Streptavidin-agarose beads (SigmaS-1638) or -pellets were washed with PBS, and 20 μg of eachbiotinylated, radiolabeled protein was added to 400 μl of beads and 400μl of PBS, vortexed for 20 seconds and centrifuged at 14,000 rpm for 5minutes. The supernatant was removed and the pellets were washed with400 μl PBS. This wash procedure was repeated twice more, and thecombined supernatants were assayed by placing them in a dosimeter versustheir respective pellets. The values are shown below in Table 4. TABLE 4Compound Supernatant Pellet orosomucoid  90%  10% biotin-oroso 7.7% 92.%asialoorosomucoid  92% 8.0% biotin-asialo  10%  90%

[0557] C. Protein-Streptavidin Binding in vivo. Biotin-asialoorosomucoidwas evaluated for the ability to couple with circulating LU-10-StrAvconjugate in vivo and to remove it from the blood. Female BALB/c mice(20-25 g) were injected i.v. with 200 μg LU-10-StrAv conjugate. Clearingagent (200 μl PBS—group 1; 400 μg non-biotinylatedasialoorosomucoid—group 2; 400 μg biotinylated asialoorosomucoid—group3; and 200 μg biotinylated asialoorosomucoid—group 4) was administeredat 25 hours following conjugate administration. A fifth group receivedPIP-I-131-LU-10-StrAv conjugate which had been saturated prior toinjection with biotin—group 5. The 400 μg dose constituted a 10:1 molarexcess of clearing agent over the initial dose of LU-10-StrAv conjugate,while the 200 μg dose constituted a 5:1 molar excess. The saturatedPIP-I-131-LU-10-StrAv conjugate was produced by addition of a 10-foldmolar excess of D-biotin to 2 mg of LU-10-StrAv followed by sizeexclusion purification on a G-25 PD-10 column.

[0558] Three mice from each group were serially bled, as describedabove, at 0.17, 1, 4 and 25 hours (pre-injection of clearing agent), aswell as at 27, 28, 47, 70 and 90 hours. Two additional animals from eachgroup were sacrificed at 2 hours post-clearing agent administration andlimited biodistributions were performed.

[0559] The blood clearance data are shown in FIG. 7. These data indicatethat circulating LU-10-StrAv radioactivity in groups 3 and 4 was rapidlyand significantly reduced, in comparison to those values obtained in thecontrol groups 1, 2 and 5. Absolute reduction in circulatingantibody-streptavidin conjugate was approximately 75% when compared tocontrols.

[0560] Biodistribution data are shown in tabular form in FIG. 8. Thebiodistribution data show reduced levels of conjugate for groups 3 and 4in all tissues except the liver, kidney and intestine, which isconsistent with the processing and excretion of radiolabel associatedwith the conjugate after complexation with biotinylatedasialoorosomucoid.

[0561] Furthermore, residual circulating conjugate was obtained fromserum samples by cardiac puncture (with the assays conducted inserum+PBS) and analyzed for the ability to bind biotin (immobilizedbiotin on agarose beads), an indicator of functional streptavidinremaining in the serum. Group 1 animal serum showed conjugate radiolabelbound about 80% to immobilized biotin. Correcting the residualcirculating radiolabel values by multiplying the remaining percentinjected dose (at 2 hours after clearing agent administration) by theremaining percent able to bind immobilize biotin (the amount ofremaining functional conjugate) leads to the graph shown in FIG. 9.Administration of 200 μg biotinylated asialoorosomucoid resulted in a50-fold reduction in serum biotin-binding capacity and, in preliminarystudies in tumored animals, has not exhibited cross-linking and removalof prelocalized LU-10-StrAv conjugate from the tumor. Removal ofcirculating targeting moiety-anti-ligand without diminishingbiotin-binding capacity at target cell sites, coupled with an increasedradiation dose to the tumor resulting from an increase in the amount oftargeting moiety-anti-ligand administered, results in both increasedabsolute rad dose to tumor and diminished toxicity to non-tumor cells,compared to what is currently-achievable using conventionalradioimmunotherapy.

[0562] A subsequent experiment was executed to evaluate lower doses ofasialoorosomucoid-biotin. In the same animal model, doses of 50, 20 and10 μg asialoorosomucoid-biotin were injected at 24 hours followingadministration of the LU-10-StrAv conjugate. Data from animals seriallybled are shown in FIG. 10, and data from animals sacrificed-twohours-after clearing agent administration are shown in FIGS. 11A (bloodclearance) and 11B (serum biotin-binding), respectively. Doses of 50 and20 μg asialoorosomucoid-biotin effectively reduced circulatingLU-10-StrAv conjugate levels by about 65% (FIG. 11A) and, aftercorrection for binding to immobilized biotin, left only 3% of theinjected dose in circulation that possessed biotin-binding capacity,compared with about 25% of the injected dose in control animals (FIG.11B). Even at low doses (approaching 1:1 stoichiometry with circulatingLU-10-StrAv conjugate), asialoorosomucoid-biotin was highly effective atreducing blood levels of circulating streptavidin-containing conjugateby an in vivo complexation that was dependent upon biotin-avidininteraction.

EXAMPLE XIV Streptavidin Anti-Ligand in Tumors

[0563] A set of female nude mice, implanted subcutaneously with LS-180human colon carcinoma xenografts as described above, were randomizedinto groups of 4 animals/timepoint. The mice were intravenously injectedwith 200 μg of 1:1 mol/mol NR-LU-10 monoclonal antibody covalentlycoupled to streptavidin (MAB-STRPT), with the conjugate formed asdescribed in Example XI above. The streptavidin portion of the conjugatewas radiolabeled with paraiodophenyl (PIP) I-125, as described inExample IV above. Groups of mice were sacrificed at 26, 30, 48, 96 and144 hours post-conjugate injection. Tissues were isolated, weighed andcounted with respect to iodine radionuclide content using conventionalprocedures therefor.

[0564] A second set of female nude mice bearing LS-180 xenografts werealso randomized into groups of 4 animals/timepoint. These mice wereintravenously injected with 50 μg of NR-LU-10 monoclonal antibodyradiolabeled with paraiodophenyl (RIP) I-131 (MAB), as described inExample IV above. Mice were sacrificed at 4, 24, 48, 128 and 168 hourspost-radiolabeled monoclonal antibody injection. Tissues were isolated,weighed and counted with respect to iodine radionuclide content usingconventional procedures therefor.

[0565] For each data set, a radioactivity standard of the injected dosewas also counted, and data were reduced to a percent of the totalinjected dose per gram of tissue. FIG. 12 shows the percent injecteddose/gram of NR-LU-10-streptavidin-PIP-I-125 and NR-LU-10-PIP-I-131 inLS-180 tumors over time. The NR-LU-10-streptavidin conjugate exhibitshigher tumor uptake and a longer retention time as compared to NR-LU-10alone.

EXAMPLE XV Streptavidin Anti-Ligand in Liver

[0566] Female nude mice xenografted with LS-180 tumor cells, asdiscussed above, were randomized into groups of 4 animals/timepoint.Mice were intravenously injected with 50 μg of biotinylated NR-LU-10monoclonal antibody that was non-covalently coupled (to form a complex)through biotin-streptavidin binding to 30 μg of streptavidin. Prior tocomplexation in vivo, the antibody portion of the complex wasradiolabeled with I-125 using chloramine-T, and the streptavidin portionwas labeled with paraiodophenyl (PIP) I-131, both of the labelingprocedures having been described above. Mice were sacrificed at 4, 24,48, 96 and 144 hours post-conjugate injection. Tissues were isolated,weighed and counted with respect to the content of each iodineradionuclide using conventional procedures therefor.

[0567] A radioactivity standard of the injected doses of each complexcomponent was also counted, and data were reduced to a percent of thetotal injected dose per gram of tissue. FIG. 13 shows the percentinjected dose per gram of streptavidin-PIP-I-131 (STREPT) andNR-LU-10-biotin-Chloramine-T-I-125 (MAB-BT) in liver over time. Thecomplex localized at the liver as a single molecule; however, theprocessing of the individual components thereof differed in the liver.The I-131-streptavidin label showed prolonged residence in the liver,while the monoclonal antibody label (I-125) was rapidly lost.

[0568] In another liver study, female nude mice xenografted withxenografted with LS-180 tumor cells, as discussed above, and wereintravenously injected with 200 μg of 1:1 mol/mol NR-LU-10 monoclonalantibody covalently coupled to streptavidin, prepared as described inExample XI above. The antibody portion of the conjugate was radiolabeledwith paraiodophenyl (PIP-I-125). Twenty four hours later, the micereceived an injection of 0.5 μg of paraiodophenyl (PIP I-131) biocytin.Mice were sacrificed at 28, 48, 120 and 168 hours post-conjugateinjection. Tissues were isolated, weighed and counted with respect tothe content of each iodine radionuclide using conventional procedurestherefor.

[0569] A radioactivity standard of the injected doses of each complexcomponent was also counted, and data were reduced to a percent of thetotal injected dose per gram of tissue (% ID/G). FIG. 14 shows thepercent injected dose per gram of streptavidin-monoclonalantibody-PIP-I-125 (STREP-MAB-I-125) and biocytin-PIP-I-131 (BT-I-131)in liver over time. When biocytin-PIP-I-131 was subsequentlyadministered, the retention of streptavidin-bound biotin radiolabel(I-131) was prolonged relative to the retention of the antibody-boundlabel (I-125) on the same moiety in the liver.

EXAMPLE XVI Tumor Uptake of PIP-Biocytin

[0570] PIP-Biocytin, as prepared and described in Example VII above, wastested to determine the fate thereof in vivo. The following data arebased on experimentation with tumored nude mice (100 mg LS-180 tumorxenografts implanted subcutaneously 7 days prior to study) thatreceived, at time 0, 200 μg of I-125 labeled NR-LU-10-Streptavidinconjugate (950 pmol), as discussed in Example XI above. At 24 hours, themice received an i.v. injection of PIP-I-131-biocytin (40 μCi) and anamount of cold carrier PIP-I-127 biocytin corresponding to doses of 42μg (69,767 pmol), 21 μg (34,884 pmol), 5.7 μg (9468 pmol), 2.85 μg (4734pmol) or 0.5 μg (830 pmol). Tumors were excised and counted forradioactivity 4 hours after PIP-biocytin injection, and the tumor uptakedata are shown in FIGS. 15A (% ID/G v. Dose) and 15B (pMOL/G v. Dose).

[0571] The three highest doses produced PIP-biocytin tumor localizationsof about 600 pmol/g. Histology conducted on tissues receiving the twohighest doses indicated that saturation of tumor-bound streptavidin wasachieved. Equivalent tumor localization observed at the 5.7 μg dose(FIG. 15B) is indicative of streptavidin saturation as well. Incontrast, the two lowest doses produced lower absolute tumorlocalization of PIP-biocytin, despite equivalent localization ofNR-LU-10-Streptavidin conjugate (tumors in all groups averaged about 40%ID/G for the conjugate).

[0572] The lowest dose group (0.5 μg) exhibited high efficiency tumordelivery of PIP-I-131-biocytin, which efficiency increased over time, asshown in FIG. 16A. A peak uptake of 85.0% ID/G was observed at the 120hour time point (96 hours after administration of PIP-biocytin). Also,the absolute amount of PIP-biocytin, in terms of % ID, showed acontinual increase in the tumor over all of the sampled time points(FIG. 16B). The decrease in uptake on a % ID/G basis (FIG. 16A) at the168 hour time point resulted from significant growth of the tumorsbetween the 120 and 168 hour time points.

[0573] In addition, FIG. 17A shows the co-localization ofNR-LU-10-Streptavidin conjugate (LU-10-StrAv) and the subsequentlyadministered PIP-Biocytin at the same tumors over time. The localizationof radioactivity at tumors by PIP-biocytin exhibited a pattern of uptakeand retention that differed from that of the antibody-streptavidinconjugate (LU-10-StrAv). LU-10-StrAv exhibited a characteristic tumoruptake pattern that is equivalent to historical studies of nativeNR-LU-10 antibody, reaching a peak value of 40% ID/G between 24 and 48hours after administration. In contrast, the PIP-Biocytin exhibited aninitial rapid accretion in the tumor, reaching levels greater than thoseof LU-10-StrAv by 24 hours after PIP-Biocytin administration. Moreover,the localization of PIP-Biocytin continued to increase out to 96 hours,when the concentration of radioactivity associated with the conjugatehas begun to decrease. The slightly greater amounts of circulatingPIP-Biocytin compared to LU-10-StrAv at these time points (shown in FIG.17B) appeared insufficient to account for this phenomenon.

[0574] As FIG. 18 clearly shows, the ratio of PIP-Biocytin toLU-10-StrAv in the tumor increased continually during the experiment,while the ratio in the blood decreased continually. This observation isconsistent with a process involving continual binding of targetingmoiety-containing conjugate (with PIP-Biocytin bound to it) from theblood to the tumor, with subsequent differential processing of thePIP-Biocytin and the conjugate. Since radiolabel associated with thestreptavidin conjugate component (compared to radiolabel associated withthe targeting moiety) has shown increased retention in organs ofmetabolic processing (Examples XIV and XV above), PIP-Biocytinassociated with the streptavidin appears to be selectively retained bythe tumor cells. Because radiolabel is retained at target cell sites, agreater accumulation of radioactivity at those sites results.

[0575] The AUC_(tumor)/AUC_(blood) for PIP-Biocytin is over twice thatof the conjugate (4.27 compared to 1.95, where AUC means “area under thecurve”). Further, the absolute AUC_(tumor) for PIP-Biocytin is nearlytwice that of the conjugate (9220 compared to 4629). Consequently, anincrease in radiation dose to tumor was achieved.

EXAMPLE XVII Polymer-Ligand Conjugation

[0576] Polylysine (approximately 10,000 Dal. molecular weight, availablefrom Sigma Chemical Co., St. Louis, Mo.) and dextran (lysine fixable,available from Sigma Chemical Co.) were derivitized with SPDP andpurified from unreacted SPDP using size exclusion chromatography (usinga PD-10 column available from Pharmacia, Piscataway, N.J.). Theresultant SPDP-derivitized adducts were reduced with DTT in pH 4.7 0.2 MNaOAc buffer to generate free reactive thiols. Reduced Tc-99m, generatedfrom stannous gluconate as described, for example, by Carlsson et al.,Biochem. J., 173: 723-737, 1978, was added. A 90% incorporation ofTc-99m was obtained for the polylysine adduct within 15 min, as measuredby ITLC. 96% of the radioactivity coeluted with the dextran using sizeexclusion (PD-10) chromatography. These results are indicative ofchelation.

EXAMPLE XVIII Preparation of Trichothecene-Linker Molecules

[0577] A. Preparation of 3-(2-Pyridinyldithio)propanoic acid. 5.00 g(52.2 mmol) of 3-mercaptopropanoic acid (Aldrich Chemical Co.,Milwaukee, Wis.) in 75 ml of dry methylene chloride was added to asolution of 5.96 g (52.2 mmol) of methoxycarbonylsulfenyl chloride(Fluka Chemika, Long Island, N.Y.) in 150 ml of dry methylene chloride.The mixture was stirred at 15-25° C. for 90 minutes and thenconcentrated. The residue was redissolved in 150 ml of dry methylenechloride and dropwise treated with 5.80 g (52.2 mmol) of2-mercaptopyridine (Aldrich Chemical Co.) in 75 ml of dry methylenechloride. The mixture was stirred at 15-25° C. for 18 hours andconcentrated to afford 11.2 g of the product as a pale yellow oil (99%).

[0578] B. Preparation of 3-(2-pyridinyldithio)propanoic acid hydrazide.Dry triethylamine (3.76 g, 37.2 mmol) was added to a solution of 8.00 g(37.2 mmol) of 3-(2-pyridinyldithio)propanoic acid in 100 ml of drytetrahydrofuran (THF). The mixture was cooled in an ice bath followed bythe addition of 5.08 g (37.2 mmol) of isobutyl chloroformate. Themixture was stirred for 5-10 minutes, and 4.92 g (37.2 mmol) oftert-butyl carbazate was added. The resultant mixture was stirred at15-25° C. for 1 hour and then concentrated. The residue was diluted with200 ml of methylene chloride and washed with water (2×100 ml). Theorganic phase was dried over magnesium sulfate, filtered andconcentrated. The residue was dissolved in ice-cold trifluoroacetic acid(160 ml) and stirred for 10 minutes after dissolution was complete. Themixture was concentrated, and the residue was chromatographed on silicagel, eluting with 85:14:1 chloroform?methanol/ammonium hydroxide, toafford 4.25 g of the product as a pale yellow solid (50%): TLC-R_(f)0.55 (85:15:1 chloroform/methanol/ammonium hydroxide).

[0579] C. Preparation of Hydrazone Derivative of 3-Dehydroanguidine and3-(2-Pyridinylithio)propanoic Acid Hydrazide. 100 microliters (0.708mmol) of trifluoroacetic anhydride at −70° C. was added to a solution of100 microliters of dry dimethylsulfoxide (1.41 mmol) in 3 ml of drymethylene chloride. The mixture was stirred at −70° C. for 10 minutesand then 70 mg (0.198 mmol) of anguidine (Sigma Chemical Co.) in 2 ml ofdry methylene chloride was added over a period of 2-3 minutes. Theresultant mixture was stirred at −70° C. for 15 minutes and then 20microliters of dry triethylamine was added. The mixture was stirred at−70° C. for 15 minutes and at 70° C. to 15° C. for 60 minutes. Theresultant mixture was diluted with 50 ml of methylene chloride andwashed with 1 N aqueous HCl (50 ml). The organic phase was dried overmagnesium sulfate, filtered and concentrated to afford crude3-dehydroanguidine. The crude material was dissolved in 3 ml of drymethanol and treated with 100 mg (0.436 mmol) of3-(2-pyridinyldithio)propanoic acid hydrazide followed by 0.021 mmol oftrifluoroacetic acid in 100 microliters of methanol. The mixture wasstirred at 15-25° C. for 5 hours and then concentrated. The residue waschromatographed on silica gel, eluting with 75% ethyl acetate/hexane, toafford 67 mg of the product, a foamy white solid, as a mixture of synand anti isomers (60%): TLC-R_(f) 0.43 and 0.60 (75% ethylacetate/hexane).

[0580] D. Preparation of a Hydrazone Derivative of 2′-Dehydro-Roridin A.

[0581] 1. Preparation of 13′-O-tert-butyldimethylsilyl-Roridin A. 100 mg(1.47 mmol) of imidazole was added to a solution of 232 mg (0.436 mmol)of Roridin A in 3 ml of dry dimethylformamide. The mixture was cooled to−5° C. and then 72 mg (0.478 mmol) of tert-butyldimethylsilyl chloride(Aldrich Chemical Co.) was added. The resultant mixture was stirred at−5° C. to 5° C. for 4 hours and then concentrated. The residue waschromatographed on silica gel, eluting first with 30% ethylacetate/hexane, next with 50% ethyl acetate/hexane and finally with 70%ethyl. acetate/hexane, to afford 137 mg of the product as a foamy whitesolid (49%): TLC-R_(f) 0.32 (30% ethyl acetate/hexane).

[0582] 2. Preparation of2′-dehydro-13′-O-tert-butyldimethylsilyl-Roridin A. At −70° C., 150microliters (1.06 mmol) of trifluoroacetic anhydride was added to asolution of 100 microliters (1.41 mmol) of dry dimethylsulfoxide in 3 mlof dry methylene chloride. The mixture was stirred at −70° C. for 15minutes and then 100 mg (0.155 mmol) of13′-O-tert-butyldimethylsilyl-Roridin A in 2 ml of dry methylenechloride was added. The resultant mixture was stirred at −70° C. for 15minutes and then 300 microliters (2.15 mmol) of dry triethylamine wasadded. This mixture was stirred at −70° C. for 30 minutes and at −70° C.to 15° C. for 30 minutes. The resultant mixture was then concentratedand the residue was chromatographed on silica gel, eluting with 30%ethyl acetate/hexane, to afford 82 mg of the product as a foamy whitesolid (82%): TLC-R_(f) 0.51 (30% ethyl acetate/hexane).

[0583] 3. Preparation of 2′-dehydro-Roridin A. 5 ml of 3:1:1 aceticacid:THF:water was added to a 10 ml round bottom flask, charged with 62mg (0.096 mmol) of 2′-dehydro-13″-O-tert-butyldimethylsilyl-Roridin A.The mixture was stirred at 45-50° C. for 4.5 hours, cooled andconcentrated. The residue was chromatographed on silica gel, elutingwith 5% methanol/methylene chloride, to afford 36 mg of the product as afoamy white solid (70%): TLC-R_(f) 0.48 (5% methanol/methylenechloride).

[0584] 4. Preparation of a hydrazone derivative of 2′-dehydro-Roridin Aand 3-(2-pyridinyldithio)propanoic acid hydrazide. To a solution of 25mg (0.471 mmol) of 2′-dehydro-Roridin A in 2 ml of dry methanol wasadded 30 mg (0.131 mmol) of 3-(2-pyridinyldithio)propanoic propanoicacid hydrazide followed by 0.013 mmol trifluoroacetic acid. The mixturewas stirred at 15-25° C. for 4 hours and then concentrated. The residuewas chromatographed on silica gel, eluting with 75% ethylacetate/hexane, to afford 25 mg of the product as a mixture of syn andanti isomers (71%): TLC-R_(f) 0.29 and 0.53 (75% ethyl acetate/hexane).

[0585] E. Preparation of a Hydrazone Derivative of2′-O-Acetyl-13′-Dehydro-Roridin A.

[0586] 1. Preparation of 2′-O-acetyl-13′-O-tert-butyldimethylsilyl-Roridin A. To a solution of 159 mg (0.246 mmol) of13′-O-tert-butyldimethylsilyl Roridin A in 4 ml of dry methylenechloride was added 200 microliters (1.43 mmol) of triethylamine, 2 mg(0.019 mmol) of dimethylaminopyridine and 120 microliters (1.27 mmol)acetic anhydride. The mixture was stirred at 15-25° C. for 16 hours andthen concentrated. The residue was chromatographed on silica gel,eluting first with 30% ethyl acetate/hexane and then with 50% ethylacetate/hexane, to afford 156 mg of the product as a foamy white solid(92%): TLC-R_(f) 0.50 (30% ethyl acetate/hexane).

[0587] 2. Preparation of 2′-O-acetyl-Roridin A. To a solution of 156 mg(0.226 mmol) of 2′-O-acetyl-13′-tert-butyldimethylsilyl-Roridin A in 3ml of dry THF was added 1.5 ml of 1M tetrabutyl ammonium fluoride. Themixture was stirred at 15-25° C. for 4 hours and then concentrated. Theresidue was chromatographed on silica gel, eluting with 50% ethylacetate/hexane, to afford 125 mg of the product as a foamy white solid(96%): TLC-R_(f) 0.17 (50% ethyl acetate/hexane).

[0588] 3. Preparation of 2′-O-acetyl-13′-dehydro-Roridin A. To asolution of 62 microliters (0.87 mmol) of dry dimethylsulfoxide in 2 mlof dry methylene chloride at −70° C. was added 62 microliters (0.439mmol) trifluoroacetic anhydride. The mixture was stirred at −70° C. for10 minutes followed by the addition of 25 mg (0.044 mmol) of2′-O-acetyl-Roridin A in 1.5 ml of dry methylene chloride over a 2-3minute period. This mixture was stirred at −70° C. for 20 minutes andthen 180 microliters (1.29 mmol) of dry triethylamine was added. Theresultant mixture was stirred at −70° C. for 15 minutes and at −70° C.to 15° C. for 20 minutes and then concentrated. The residue waschromatographed on silica gel, eluting with 5% methanol/methylenechloride, to afford 21 mg of the product as a foamy white solid.

[0589] 4. Preparation of a hydrazone derivative of2′-acetyl-13′-dehydro-Roridin A and 3-(2-pyridinyldithio)propanoic acidhydrazide. To a solution of 21 mg (0.037 mmol) of2′-O-acetyl-13′-dehydro-Roridin A in 3 ml of dry methanol was added 25mg (0.11 mmol) of 3-(2-pyridinyldithio)propanoic acid hydrazide followedby 0.011 mmol of trifluoroacetic acid in 100 microliters of drymethanol. The mixture was stirred at 15-25° C. for 3 hours and thenconcentrated. The residue was chromatographed on silica gel, elutingfirst with 75% ethyl acetate/hexane and then with ethyl acetate, toafford 18 mg of the product as a colorless oil (62%): TLC-R_(f) 0.30(75% ethyl acetate/hexane).

[0590] F. Preparation of2′-Desoxy-2′-alpha-(N-hydroxysuccinimidyl-3-dithiopropanoicacid)-Roridin A.

[0591] 1. Preparation of2′-desoxy-2′-beta-iodo-13′-O-tert-butyldimethylsilyl-Roridin A. To asolution of 99 mg (0.153 mmol) of 13′-O-tert-butyldimethylsilyl-RoridinRoridin A in 5 ml of dry methylene chloride, at 0° C., was added 80microliters (0.459 mmol) of dry diiso-propylethylamine followed by 51microliters (0.303 mmol) of trifluoromethanesulfonic anhydride. Themixture was stirred at 0° C. for 30 minutes and then diluted with 30 mlof methylene chloride and washed with 20 ml of 1N aqueous HCl. Theorganic phase was dried over magnesium sulfate, filtered andconcentrated. The residue was diluted with 5 ml of acetone and then 235mg (1.72 mmol) of sodium iodide. The mixture was stirred at reflux for20 minutes and then diluted with 50 ml of methylene chloride and washedwith 25 ml of water. The organic phase was dried over magnesium sulfate,filtered and concentrated. The residue was chromatographed on silicagel, eluting with 30% ethyl acetate/hexane to afford 95 mg of theproduct as a foamy white solid (87%): TLC-R_(f) 0.55 (35% ethylacetate/hexane).

[0592] 2. Preparation of 2′-desoxy-2′-beta-iodo-Roridin A. To 95 mg(0.126 mmol) of2′-desoxy-2′-beta-iodo-13′-O-tert-butyldimethylsilyl-Roridin A was added5 ml of 3:1:1 acetic acid:THF:water. The mixture was stirred at 60-65°C. for 4 hours and then concentrated. The residue was chromatographed onsilica gel, eluting with 60% ethyl acetate/hexane, to afford 67 mg ofthe product as a foamy white solid (83%).

[0593] 3. Preparation of 2′-desoxy-2′-alpha-thioacetyl-Roridin A. To asolution of 67 mg (0.104 mmol) of 2′-desoxy-2′-beta-iodo-Roridin A in 5ml of absolute ethanol was added 240 mg (2.10 mmol) of potassiumthioacetate. The mixture was stirred at 65-70° C. for 5 hours, cooled,diluted with 50 ml of methylene chloride and washed with 50 ml of water.The organic phase was dried over magnesium sulfate, filtered andconcentrated. The residue was chromatographed on silica gel, elutingwith 60% ethyl acetate/hexane, to afford 58 mg of the product as a foamywhite solid (94%): TLC-R_(f) 0.53 (60% ethyl acetate/hexane).

[0594] 4. Preparation of 2′-desoxy-2′-alpha-mercaptyl-Roridin A. To asolution of 57 mg (0.097 mmol) of 2′-desoxy-2′-alpha-thioacetyl-RoridinA in 4 ml of ethanol was added 2 ml of 28% aqueous ammonium hydroxide.The mixture was stirred at 65-70° C. for 6 hours. The mixture wasconcentrated and the residue was chromatographed on silica gel, elutingwith 10% ethyl acetate/hexane, to afford 25 mg of an intermediatedisulfide. The disulfide was diluted into 3 ml of methylene chloride and0.5 ml of methanol and 25 microliters (0.10 mmol) of tributylphosphinewas added. The mixture was stirred at 15-25° C. for 15 minutes and thenconcentrated. The residue was chromatographed on silica gel, elutingwith 60% ethyl acetate/hexane, to afford 23 mg of the product as acolorless oil (43%): TLC-R_(f) 0.39 (60% ethyl acetate/hexane).

[0595] 5. Preparation of 2′-desoxy-2′-alpha-(3-dithiopropanoicacid)-Roridin A. To a solution of 21 mg (0.038 mmol) of2′-desoxy-2′-alpha-mercaptyl-Roridin A in 3 ml of ethanol was added 20mg (0.093 mmol) of 3-(2-pyridinyldithio)propanoic acid followed by 25microliters (0.179 mmol) of triethylamine. The mixture was stirred at15-25° C. for 30 minutes and then diluted with 50 ml of ethyl acetateand washed with 10 ml of 1N aqueous HCl. The organic phase was driedover magnesium sulfate, filtered and concentrated. The residue waschromatographed on silica gel, eluting first with 0.1% acetic acid/ethylacetate and then with 0.1:5:95 acetic acid:methanol:ethyl acetate, toafford 22 mg of the product as a near colorless oil (88%): TLC-R_(f)0.37 (0.1:5:95 acetic acid:methanol:ethyl acetate).

[0596] 6. Preparation of 2′-desoxy-2′-alpha-(N-hydroxysuccinimidyl-3-dithiopropanoic acid)-Roridin A. To a solution of 22 mg(0.0337 mmol) of 2′-desoxy-2′-alpha-(3-dithiopropanoic acid)-Roridin Ain 2 ml of dry THF was added 12 mg (0.104 mmol) of N-hydroxysuccinimidefollowed by 18 mg (0.087 mmol) of dicyclohexyl carbodiimide. The mixturewas stirred at 15-25° C. for 4 hours and then 20 microliters of aceticacid was added. The mixture was stirred an additional 30 minutes andthen filtered through a plug of glass wool. The solids were washed with3 ml of THF. The filtrates were combined and concentrated. The residuewas chromatographed on silica gel, eluting first with 0.1:50:50 aceticacid:ethyl acetate:hexane and then with 0.1% acetic acid/ethyl acetate,to afford 18.mg of the product as a white solid (71%): TLC-R_(f) 0.69(ethyl acetate).

EXAMPLE XIX Preparation of Trichothecene-Containing Conjugates

[0597] Conjugation of trichothecenes as described in the specificationmay be conducted through either of two exemplary synthesis schemes: (1)reaction of the amino groups of a targeting moiety with N-hydroxylsuccinimidate (NHS)-derivatized trichothecenes or (2) the reaction ofreduced disulfides or free sulfhydryls of a targeting moiety with2-pyridinyl dithio-derivatized trichothecenes.

[0598] A. Antibody Conjugation of NHS-Derivatized Trichothecenes. Thisconjugation was generally performed in 6.2 mM sodium borate buffer (0.5M, pH 8), containing the antibody stabilizer molecusol at 1% wt/vol. TheNHS-derivatized trichothecene dissolved in 100% DMSO was added dropwiseto a stirred solution of antibody at a final antibody concentration of1.0 mg/ml reaction solution. To achieve a trichothecene loading of6-9/antibody, NHS-derivatized trichothecene was offered at a 30:1trichothecene:antibody molar ratio at a final concentration of 25% DMSOvol/vol. The reaction mixture was incubated with continuous stirring atroom temperature for 1 hour prior to purification by size exclusionchromatography, extensive dialysis, concentration via membranecentrifugation and sterile filtration. Conjugates thus prepared werestored refrigerated (e.g., at 5-10° C.) or quick frozen in liquidnitrogen and then stored at −70° C. until use).

[0599] Specifically for succinimidyl-2′-Roridin A 3-dithiopropionateconjugation to antibody, 0.40 ml of 6.2 mM sodium borate buffer (0.5 M,pH 8.0) and 0.20 ml of 100 mg molecusol/ml H₂O were added to 1.0 ml of2.0 mg NR-LU-10/ml in PBS (i.e., 6.2 mM sodium phosphate, 150 mM NaCl,pH 7.2). 0.50 ml of DMSO having 290 micrograms ofsuccinimidyl-2′-Roridin A 3-dithiopropionate dissolved therein (for a30:1 trichothecene:antibody molar offering ratio) was added dropwise andwith stirring over a 10 second time period. To purify the resultantproduct, 1 ml aliquots of reaction solution were applied to PD-10Sephadex columns (Pharmacia, Uppsala, Sweden) equilibrated in PBScontaining 1% molecusol wt/vol. The eluted conjugate was collected inthe 2.4-4.8 ml fraction. The PD-10 purified aliquots were then pooled,exhaustively dialyzed against PBS containing 1% molecusol (wt/vol) usingSpectra/POR molecular porous membrane tubing (Spectrum MedicalIndustries, Inc., Los Angeles, Calif.), concentrated on a CentriconPM-30 Microconcentrator (Amicon Div., W.R. Grace & Co., Beverly, Mass.),and sterile filtered using a 2.2 micron Acrodisc (Gelman Sciences., AnnArbor, Mich.). Conjugates thus prepared were stored refrigerated (e.g.,at 5-10° C.) or quick frozen in liquid nitrogen and then stored at −70°C. until use).

[0600] B. Antibody Conjugation of 2-Pyridinyl-Dithio-DerivatizedTrichothecenes. In conjugation of 2-pyridinyl-dithio-derivatizedtrichothecenes, NR-LU-10 disulfides were first reduced by incubatingNR-LU-10 at 5-10 mg in 1 ml of PBS containing 10-15 mM dithiothreitolfor 15 minutes at room temperature. The reduced product was purifiedover a PD-10 column that had been equilibrated in degassed 0.1 M sodiumphosphate, pH 7.5, containing 0.1 M NaCl, 1% molecusol wt/vol and 0.2 mMEDTA (Na salt). Immediately after reduced-disulfide NR-LU-10purification, dropwise addition of 2-pyridinyl-dithio-derivatizedtrichothecene dissolved in DMSO (0.40 ml of DMSO containing 432micrograms of 3-(2-pyridinyldithio)propionate hydrazide 2′-Roridin-Ahydrazone or containing 337 micrograms of3-(2-pyridinyldithio)propionate hydrazide 3-anguidine hydrazone orcontaining 459 micrograms of 3-(2-pyridinyldithio)propionate hydrazide2′-acetyl-13′-Roridin A hydrazone) to 2 mg of the reduced disulfideNR-LU-10 solution (a 44:1 trichothecene:antibody molar offering ratio)was conducted with stirring. Generally in conducting the conjugation,the reaction concentration of antibody was 0.8-1.2 mg/ml, DMSO was16-25% vol/vol, and trichothecene to antibody molar offering ranged from40:1 to 50:1 (in order to achieve a loading of approximately 6trichothecenes per antibody). After 1 hour of incubation at roomtemperature, purification proceeded analogously to the purificationscheme described in Example XVII(A) above. Conjugates thus prepared werestored refrigerated (e.g., at 5-10° C.) or quick frozen in liquidnitrogen and then stored at −70° C. until use).

[0601] C. Trichothecene-Polymer-Ligand Conjugation.

[0602] Experimentation involving the use of abiotin-dextran-trichothecene conjugate in a pretargeting approachincluded:

[0603] trace labeling using an I-125 PIP NHS ester of an availablelysine of a 70,000 dalton dextran molecule that had been biotinylated inaccordance with techniques discussed herein in Example XVII, yieldingradiotagged dextran biotin (represented as dextran*-biotin);

[0604] trichothecene drug conjugation to the remaining lysines using anNHS activated trichothecene;

[0605] demonstration of binding of the drug-dextran*-biotin molecule toimmobilized avidin; and

[0606] assessment of serum clearance in mice.

[0607] Biotinylated dextran having a molecular weight of 70,000 daltons,with 18 moles of biotin covalently bound thereto and 18 additionallysine epsilon amino groups, was purchased from Sigma Chemical Co. (St.Louis, Mo.). To radiolabel the material, 4 mC of I-125 PIP NHS ester ofspecific activity of 2200 mCi/mmole (New England Nuclear, Boston, Mass.)in acetonitrile in a 2 ml glass vial was blown down to dryness in anitrogen stream. 10 mg of biotinylated, lysine-derivatized dextranlyophilizate, reconstituted with 0.650 ml of 1.0M sodium borate, pH 9.0,was added to the iodinating compound and incubated at room temperaturefor 10 minutes. Following reaction, the radioiodinated dextran moietywas purified by size exclusion chromatography using a PD-10 column(Pharmacia, Uppsala, Sweden) equilibrated in phosphate buffered saline(i.e., 6.2M sodium phosphate, 150 mM NaCl, pH.7.2) containing 1%molecusol (Pharmatec, Alachua, Fla.). The biotin-dextran* eluted fromthe column in the 2.4-4.8 ml fractions at a specific activity of 0.2mCi/mg and a concentration of 3.5 mg/ml.

[0608] To derivatize the biotin-dextran* with trichothecene, 0.9 ml ofbiotin-dextran* was diluted with 1.3 ml of sodium borate buffer, 0.3M,Ph 8.5, containing 1% molecusol followed by addition, with stirring of1.2 ml of DMSO containing 1.2 mg of2′-Desoxy-2′-alpha-(N-hydroxysuccinimidyl-3-dithiopropanoicacid)-Roridin A (i.e., 2:1 drug to available lysine molar ratio). Afterincubation for 1.5 hours at room temperature, each 1 ml aliquot ofreaction mixture was purified as noted above with a PD-10 columnequilibrated in PBS. Yields exceeded 85%.

[0609] To establish that the biotin-dextran*-trichothecene molecule wasable to bind to avidin or streptavidin, 1 microgram of biotin-dextran*and 1 microgram of biotin-dextran*-trichothecene were incubated for 15minutes at room temperature with 1 unit of avidin insolubilized onagarose beads (Sigma Chemical Co., St. Louis, Mo.) in 0.2 ml of 0.2 M Pibuffer, pH 6.3 containing 150 mM NaCl. Following this incubation, thepercent radioactivity bound to the agarose beads was assessed afterdilution with 1.4 ml buffer, centrifugation of the agarose suspensionand three washings of the pellets with 1.4 ml buffer. 100% binding wasobserved for both biotin-dextran* and biotin-dextran*-trichothecene.

[0610] Serum clearance studies of biotin-dextran* andbiotin-dextran*-trichothecene were also performed in Balb C mice. Serialblood samplings revealed that he two molecules exhibited substantiallysimilar serum clearance upon injection of 2 μCi thereof.

EXAMPLE XXI Synthesis of DOTA-Biotin Conjugates

[0611] A. Synthesis of Nitro-Benzyl-DOTA.

[0612] The synthesis of aminobenzyl-DOTA was conducted substantially inaccordance with the procedure of McMurry et al., Bioconjugate Chem., 3:108-117, 1992. The critical step in the prior art synthesis is theintermolecular cyclization between disuccinimidylN-(tert-butoxycarbonyl)iminodiacetate and N-(2-aminoethyl)-4-nitrophenylalaninamide to prepare1-(tert-butoxycarbonyl)-5-(4-nitrobenzyl)-3,6,11-trioxo-1,4,7,10-tetraazacyclododecane.In other words, the critical step is the intermolecular cyclizationbetween the bis-NHS ester and the diamine to give the cyclized dodecane.McMurry et al. conducted the cyclization step on a 30 mmol scale,dissolving each of the reagents in 100 ml DMF and adding via a syringepump over 48 hours to a reaction pot containing 4 liters dioxane.

[0613] A 5− scale-up of the McMurry et al. procedure was not practicalin terms of reaction volume, addition rate and reaction time. Processchemistry studies revealed that the reaction addition rate could besubstantially increased and that the solvent volume could be greatlyreduced, while still obtaining a similar yield of the desiredcyclization product. Consequently on a 30 mmol scale, each of thereagents was dissolved in 500 ml DMF and added via addition funnel over27 hours to a reaction pot containing 3 liters dioxane. The additionrate of the method employed involved a 5.18 mmol/hour addition rate anda 0.047 M reaction concentration.

[0614] B. Synthesis of a D-alanine-linked conjugate with a preservedbiotin carboxy moiety. A reaction scheme to form a compound of thefollowing formula is discussed below.

[0615] The D-alanine-linked conjugate was prepared by first couplingD-alanine (Sigma Chemical Co.) to biotin-NHS ester. The resultantbiotinyl-D-alanine was then activated with1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride (EDCI) andN-hydroxysuccinimide (NHS). This NHS ester was reacted in situ withDOTA-aniline to give the desired product which was purified bypreparative HPLC.

[0616] More specifically, a mixture of D-alanine (78 mg, 0.88 mmol, 1.2equivalents), biotin-NHS ester (250 mg, 0.73 mmol, 1.0 equivalent),triethylamine (0.30 ml, 2.19 mmol, 3.0 equivalents) in DMF (4 ml) washeated at 110° C. for 30 minutes. The solution was cooled to 23° C. andevaporated. The product solid was acidified with glacial acetic acid andevaporated again. The product biotinyl-D-alanine, a white solid, wassuspended in 40 ml of water to remove excess unreacted D-alanine, andcollected by filtration. Biotinyl-D-alanine was obtained as a whitesolid (130 mg, 0.41 mmol) in 47% yield.

[0617] NHS (10 mg, 0.08 mmol) and EDCI (15 mg, 0.07 mmol) were added toa solution of biotinyl-D-alanine (27 mg, 0.08 mmol) in DMF (1 ml). Thesolution was stirred at 23° C. for 60 hours, at which time TLC analysisindicated conversion of the carboxyl group to the N-hydroxy succinimidylester. Pyridine (0.8 ml) was added followed by DOTA-aniline (20 mg, 0.04mmol). The mixture was heated momentarily at approximately 100° C., thencooled to 23° C. and evaporated. The product,DOTA-aniline-D-alanyl-biotinamide was purified by preparative HPLC.

[0618] C. Synthesis of N-hydroxyethyl-linked conjugate.

[0619] Iminodiacetic acid dimethyl ester is condensed withbiotin-NHS-ester to give biotinyl dimethyl iminodiacetate. Hydrolysiswith one equivalent of sodium hydroxide provides the monomethyl esterafter purification from under and over hydrolysis products. Reduction ofthe carboxyl group with borane provides the hydroxyethyl amide. Thehydroxyl group is protected with t-butyl-dimethyl-silylchloride. Themethyl ester is hydrolysed, activated with EDCI and condensed withDOTA-aniline to form the final product conjugate.

[0620] D. Synthesis of N-Me-LC-DOTA-biotin. A reaction scheme is shownbelow.

[0621] Esterification of 6-Aminocaproic acid (Sigma Chemical Co.) wascarried out with methanolic HCl. Trifluoroacetylation of the amino groupusing trifluoroacetic anhydride gaveN-6-(methylcaproyl)-trifluoroacetamide. The amide nitrogen wasmethylated using sodium hydride and iodomethane in tetrahydrofuran. Thetrifluoroacetyl protecting group was cleaved in acidic methanol to givemethyl 6-methylamino-caproate hydrochloride. The amine was condensedwith biotin-NHS ester to give methyl N-methyl-caproylamido-biotin.Saponification afforded the corresponding acid which was activated withEDCI and NHS and, in situ, condensed with DOTA-aniline to giveDOTA-benzylamido-N-methyl-caproylamido-biotin.

[0622] 1. Preparation of methyl 6-aminocaproate hydrochloride. Hydrogenchloride (gas) was added to a solution of 20.0 g (152 mmol) of6-aminocaproic acid in 250 ml of methanol via rapid bubbling for 2-3minutes. The mixture was stirred at 15-25° C. for 3 hours and thenconcentrated to afford 27.5 g of the product as a white solid (99%):

[0623] H-NMR (DMSO) 9.35 (1 H, broad t), 3.57 (3H, s), 3.14 (2H,quartet), 2.28 (2H, t), 1.48 (4H, multiplet), and 1.23 ppm (2H,multiplet).

[0624] 2. Preparation of N-6-(methylcaproyl)-trifluoroacetamide. To asolution of 20.0 g (110 mmol) of methyl 6-aminocaproate hydrochloride in250 ml of dichloromethane was added 31.0 ml (22.2 mmol) oftriethylamine. The mixture was cooled in an ice bath and trifluoroaceticanhydride (18.0 ml, 127 mmol) was added over a period of 15-20 minutes.The mixture was stirred at 0-10° C. for 1 hour and concentrated. Theresidue was diluted with 300 ml of ethyl acetate and saturated aqueoussodium bicarbonate (3×100 ml). The organic phase was dried overanhydrous magnesium sulfate, filtered and concentrated to afford 26.5 gof the product as a pale yellow oil (100%):

[0625] H-NMR (DMSO) 3.57 (3H, s), 3.37 (2H, t), 3.08 (1.9H, quartet,N—CH₃), 2.93 (1.1H, s, N—CH₃), 2.30 (2H, t), 1.52 (4H, multiplet), and1.23 ppm (2H, multiplet).

[0626] 3. Preparation of methyl 6-N-methylamino-caproate hydrochloride.To a solution of 7.01 g (29.2 mmol) ofN-6-(methylcaproyl)-trifluoroacetamide in 125 ml of anhydroustetrahydrofuran was slowly added 1.75 g of 60% sodium hydride (43.8mmol) in mineral oil. The mixture was stirred at 15-25° C. for 30minutes and then 6.2 g (43.7 mmol) of iodomethane was added. The mixturewas stirred at 15-25° C. for 17 hours and then filtered through celite.The solids were rinsed with 50 ml of tetrahydrofuran. The filtrates werecombined and concentrated. The residue was diluted with 150 ml of ethylacetate and washed first with 5% aqueous sodium sulfite (2×100 ml) andthen with 100 ml of 1 N aqueous hydrochloric acid. The organic phase wasdried over anhydrous magnesium sulfate, filtered and concentrated toafford a yellow oily residue. The residue was diluted with 250 ml ofmethanol and then hydrogen chloride (gas) was rapidly bubbled into themixture for 2-3 minutes. The resultant mixture was refluxed for 18hours, cooled and concentrated. The residue was diluted with 150 ml ofmethanol and washed with hexane (3×150 ml) to remove mineral oilpreviously introduced with NaH. The methanol phase was concentrated toafford 4.91 g of the product as a yellow oil (86%):

[0627] H-NMR (DMSO) 8.80 (2H, broad s), 3.58 (3H, s), 2.81 (2H,multiplet), 2.48 (3H, s), 2.30 (2H, t), 1.52 (4H, multiplet), and 1.29ppm (2H, multiplet).

[0628] 4. Preparation of methyl 6-(N-methylcaproylamido-biotin.N-hydroxysuccinimidyl biotin (398 mg, 1.16 mmol) was added to a solutionof methyl 6-(N-methyl)aminocaproate hydrochloride. (250 mg, 1.28 mmol)in DMF (4.0 ml) and triethylamine (0.18 ml, 1.28 mmol). The mixture washeated in an oil bath at 100° C. for 10 minutes. The solution wasevaporated, acidified with glacial acetic acid and evaporated again. Theresidue was chromatographed on a 25 mm flash chromatography columnmanufactured by Ace Glass packed with 50 g silica (EM Science,Gibbstown, N.J., particle size 0.40-0.63 mm) eluting with 15%MeOH/EtOAc. The product was obtained as a yellow oil (390 mg) in 79%yield.

[0629] 5. Preparation of 6-(N-methyl-N-biotinyl)amino caproic acid. To asolution of methyl 6-(N-methyl-caproylamido-biotin (391 mg, 1.10 mmol)in methanol (2.5 ml) was added a 0.95 N NaOH solution (1.5 ml). Thissolution was stirred at 23° C. for 3 hours. The solution was neutralizedby the addition of 1.0 M HCl (1.6 ml) and evaporated. The residue wasdissolved in water, further acidified with 1.0 M HCl (0.4 ml) andevaporated. The gummy solid residue was suspended in water and agitatedwith a spatula until it changed into a white powder. The powder wascollected by filtration with a yield of 340 mg.

[0630] 6. Preparation of DOTA-benzylamido-N-methyl-caproylamido-biotin.A suspension of 6-(N-methyl-N-biotinyl)amino caproic acid (29 mg, 0.08mmol) and N-hydroxysuccinimide (10 mg, 0.09 mmol) in DMF (0.8 ml) washeated over a heat gun for the short time necessary for the solids todissolve. To this heated solution was added EDCI (15 mg, 0.08 mmol). Theresultant solution was stirred at 23° C. for 20 hours. To this stirredsolution were added aminobenzyl-DOTA (20 mg, 0.04 mmol) and pyridine(0.8 ml). The mixture was heated over a heat gun for 1 minute. Theproduct was isolated by preparative HPLC, yielding 3 mg.

[0631] E. Synthesis of a bis-DOTA conjugate with a preserved biotincarboxy group. A reaction scheme is shown below.

[0632] 1. Preparation of methyl 6-bromocaproate (methyl6-bromohexanoate). Hydrogen chloride (gas) was added to a solution-of5.01 g (25.7 mmol) of 6-bromocaproic acid in 250 ml of methanol viavigorous bubbling for 2-3 minutes. The mixture was stirred at 15-25° C.for 3 hours and then concentrated to afford 4.84 g of the product as ayellow oil (90%):

[0633] H-NMR (DMSO) 3.58 (3H, s), 3.51 (2H, t), 2.29 (2H, t), 1.78 (2H,pentet), and 1.62-1.27 ppm (4H, m).

[0634] 2. Preparation of N,N-bis-(methyl 6-hexanoyl)-aminehydrochloride. To a solution of 4.01 g (16.7 mmol) of N-(methyl6-hexanoyl)-trifluoroacetamide (prepared in accordance with section D.2.herein) in 125 ml of anhydrous tetrahydrofuran was added 1.0 g (25 mmol)of 60% sodium hydride in mineral oil. The mixture was stirred at 15-25°C. for 1 hour and then 3.50 g (16.7 mmol) of methyl 6-bromocaproate wasadded and the mixture heated to reflux. The mixture-was stirred atreflux for 22 hours. NMR assay of an aliquot indicated the reaction tobe incomplete. Consequently, an additional 1.00 g (4.8 mmol) of methyl6-bromocaproate was added and the mixture stirred at reflux for 26hours. MNR assay of an aliquot indicated the reaction to be incomplete.An additional 1.0 g of methyl 6-bromocaproate was added and the mixturestirred at reflux for 24 hours. NMR assay of an aliquot indicated thereaction to be near complete. The mixture was cooled and then directlyfiltered through celite. The solids were rinsed with 100 ml oftetrahydrofuran. The filtrates were combined and concentrated. Theresidue was diluted with 100 ml of methanol and washed with hexane(3×100 ml) to remove the mineral oil introduced with the sodium hydride.The methanol phase was treated with 6 ml of 10 N aqueous sodiumhydroxide and stirred at 15-25° C. for 3 hours. The mixture wasconcentrated. The residue was diluted with 100 ml of deionized water andacidified to pH 2 with concentrated HCl. The mixture was washed withether (3×100 ml). The aqueous phase was concentrated, diluted with 200ml of dry methanol and then hydrogen chloride gas was bubbled throughthe mixture for 2-3 minutes. The mixture was stirred at 15-25° C. for 3hours and then concentrated. The residue was diluted with 50 ml of drymethanol and filtered to remove inorganic salts. The filtrate wasconcentrated to afford 1.98 g of the product as a white solid (38%):

[0635] H-NMR (DMSO) 8.62 (2H, m) 3.58 (6H, s), 2.82 (4H, m) 2.30 (4H,t), 1.67-1.45 (8H, m) and 1.38-1.22 ppm (4H, m).

[0636] 3. Preparation of N,N=bis-(methyl 6-hexanoyl)-biotinamide. To asolution of 500 mg (1.46 mmol) of N-hydroxysuccinimidyl biotin in 15 mlof dry dimethyl-formamide was added 600 mg (1.94 mmol) ofN,N-bis-(methyl 6-hexanoyl)amine hydrochloride followed by 1.0 ml oftriethylamine. The mixture was stirred at 80-85° CC. for 3 hours andthen cooled and concentrated. The residue was chromatographed on silicagel, eluting with 20% methanol/ethyl acetate, to afford 620 mg of theproduct as a near colorless oil (85%):

[0637] H-NMR (CDCl₃) 5.71 (1H, s), 5.22 (1H, s), 4.52 (1H, m), 4.33 (1H,m), 3.60 (3H, s), 3.58 (3H, s), 3.34-3.13 (5H, m), 2.92 (1H, dd), 2.75(1H, d), 2.33 (6H, m) and 1.82-1.22 ppm (18H, m); TLC-R_(f) 0.39 (20:80methanol/ethyl acetate).

[0638] 4. Preparation of N,N-bis-(6-hexanoyl)-biotinamide. To a solutionof 610 mg (0.819 mmol) of N,N-bis-(methyl 6-hexanoyl)-biotinamide in 35ml of methanol was added 5.0 ml of 1N aqueous sodium hydroxide. Themixture was stirred at 15-25° C. for 4.5 hours and then concentrated.The residue was diluted with 50 ml of deionized water acidified to pH 2with 1N aqueous hydrochloric acid at 4° C. The product, whichprecipitated out as a white solid, was isolated by vacuum filtration anddried under vacuum to afford 482 mg (84%):

[0639] H-NMR (DMSO) 6.42 (1H, s), 6.33 (1H, s), 4.29 (1H, m), 4.12 (1H,m), 3.29-3.04 (5H, m), 2.82 (1H, dd), 2.57 (1H, d), 2.21 (6H, m) and1.70-1.10 ppm (18H, m).

[0640] 5. Preparation of N′,N′-bis-(N-hydroxy-succinimidyl6-hexanoyl)-biotinamide. To a solution of 220 mg (0.467 mmol) ofN,N-bis-(6-hexanoyl)-biotinamide in 3 ml of dry dimethylformamide wasadded 160 mg (1.39 mmol) of N-hydroxysuccinimide followed by 210 mg(1.02 mmol) of dicyclohexyl-carbodiimide. The mixture was stirred at15-25° C. for 17 hours and then concentrated. The residue waschromatographed on silica gel, eluting with 0.1:20:80 aceticacid/methanol/ethyl acetate, to afford 148 mg of the product as a foamyoff-white solid (48%):

[0641] H-NMR (DMSO) 6.39 (1H, s), 6.32 (1H, s), 4.29 (1H, m), 4.12 (1H,m), 3.30-3.03 (5H, m), 2.81 (9H, dd and s), 2.67 (4H, m), 2.57 (1H, d),2.25 (2H, t), 1.75-1.20 (18H, m); TLC-R_(f) 0.37 (0.1:20:80 aceticacid/methanol/ethyl acetate).

[0642] 6. Preparation ofN,N-bis-(6-hexanoylamidobenzyl-DOTA)-biotinamide. To a mixture of 15 mgof DOTA-benzylamine and 6.0 mg of N′,N′-bis-(N-hydroxy-succinimidyl6-hexanoyl)-biotinamide in 1.0 ml of dry dimethylformamide was added 0.5ml of dry pyridine. The mixture was stirred at 45-50° C. for 4.5 hoursand at 15-25° C. for 12 hours. The mixture was concentrated and theresidue chromatographed on a 2.1×2.5 cm octadecylsilyl (ODS)reverse-phase preparative HPLC column eluting with a −−20 minutegradient profile of 0.1:95:5 to 0.1:40:60 trifluoroaceticacid:water:acetonitrile at 13 ml/minute to afford the desired product.The retention time was 15.97 minutes using the aforementioned gradientat a flow rate of 1.0 ml/minute on a 4.6 mm×25 cm ODS analytical HPLCcolumn.

[0643] F. Synthesis of an N-methyl-glycine linked conjugate. A reactionscheme for this synthesis is shown below.

[0644] The N-methyl glycine-linked DOTA-biotin conjugate was prepared byan analogous method to that used to prepare D-alanine-linked DOTA-biotinconjugates. N-methyl-glycine (trivial name sarcosine, available fromSigma Chemical Co.) was condensed with biotin-NHS ester in DMF andtriethylamine to obtain N-methyl glycyl-biotin. N-methyl-glycyl biotinwas then activated with EDCI and NHS. The resultant NHS ester was notisolated and was condensed in situ with DOTA-aniline and excesspyridine. The reaction solution was heated at 60° C. for 10 minutes andthen evaporated. The residue was purified by preparative HPLC to give[(N-methyl-N-biotinyl)-N-glycyl]-aminobenzyl-DOTA.

[0645] 1. Preparation of (N-methyl)glycyl biotin. DMF (8.0 ml) andtriethylamine (0.61 ml, 4.35 mmol) were added to solids N-methyl glycine(182 mg, 2.05 mmol) and N-hydroxy-succinimidyl biotin (500 mg, 1.46mmol). The mixture was heated for 1 hour in an oil bath at 85° C. duringwhich time the solids dissolved producing a clear and colorlesssolution. The solvents were then evaporated. The yellow oil residue wasacidified with glacial acetic acid, evaporated and chromatographed on a27 mm column packed with 50 g silica, eluting with 30% MeOH/EtOAc 1%HOAc to give the product as a white solid (383 mg) in 66% yield.

[0646] H-NMR (DMSO): 1.18-1.25 (m, 6H, (CH₂)₃), 2.15, 2.35 (2 t's, 2H,CH₂CO), 2.75 (m, 2H, SCH₂), 2.80, 3.00 (2 s's, 3H, NCH₃), 3.05-3.15 (m,1H, SCH), 3.95, 4.05 (2 s's, 2H, CH₂N), 4.15, 4.32 (2 m's, 2H, 2CHN's),6.35 (s, NH), 6.45 (s, NH).

[0647] 2. Preparation of [(N-methyl-N-biotinyl)glycyl]aminobenzyl-DOTA.N-hydroxysuccinimide (10 mg, 0.08 mmol) and EDCI (15 mg, 6.08 mmol) wereadded to a solution of (N-methylglycyl biotin (24 mg, 0.08 mmol) in DMF(1.0 ml). The solution was stirred at 23° C. for 64 hours. Pyridine (0.8ml) and aminobenzyl-DOTA (20 mg, 0.04 mmol) were added. The mixture washeated in an oil bath at 63° C. for 10 minutes, then stirred at 23° C.for 4 hours. The solution was evaporated. The residue was purified bypreparative HPLC to give the product as an off white solid (8 mg, 0.01mmol) in 27% yield.

[0648] H-NMR (D₂O): 1.30-1.80 (m, 6H), 2.40, 2.55 (2 t's, 2H, CH₂CO),2.70-4.2 (complex multiplet), 4.35 (m, CHN), 4.55 (m, CHN), 7.30 (m, 2H,benzene hydrogens), 7.40 (m, 2H, benzene hydrogens).

[0649] G. Synthesis of a short chain amine-linked conjugate with areduced biotin carboxy group. A two-part reaction scheme is shown below.

[0650] The biotin carboxyl group is reduced with diborane in THF to givea primary alcohol. Tosylation of the alcohol with tosyl chloride inpyridine affords the primary tosylate. Aminobenzyl DOTA is acylated withtrifluoroacetic anhydride in pyridine to give(N-trifluoroacetyl)aminobenzyl-DOTA. Deprotonation with 5.0 equivalentsof sodium hydride followed by displacement of the biotin tosylateprovides the(N-trifluoracetamido-N-descarboxylbiotinyl)aminobenzyl-DOTA. Acidiccleavage of the N-trifluoroacetamide group with HCl(g) in methanolprovides the amine-linked DOTA-biotin conjugate.

EXAMPLE XXII Clearing Agent Evaluation Experimentation

[0651] The following experiments conducted on non-tumor-bearing micewere conducted using female BALB/c mice (20-25 g). For tumor-bearingmice experimentation, female nude mice were injected subcutaneously withLS-180 tumor cells, and, after 7 d, the mice displayed 50-100 mg tumorxenografts. The monoclonal antibody used in these experiments wasNR-LU-10. When radiolabeled, the NR-LU-10-streptavidin conjugate wasradiolabeled with I-125 using procedures described herein. Whenradiolabeled, PIP-biocytin was labeled with I-131 or I-125 usingprocedures described herein.

[0652] A. Utility of Asialoorosomucoid-Biotin (AO-Bt) in ReducingCirculating Radioactivity from a Subsequently Administered RadiolabeledBiotin Ligand. Mice bearing LS-180 colon tumor xenografts were injectedwith 200 micrograms NR-LU-10 antibody-streptavidin (MAb-StrAv) conjugateat time 0, which was allowed to prelocalize to tumor for 22 hours. Atthat time, 20 micrograms of AO-Bt was administered to one group ofanimals. Two hours later, 90 micrograms of a radioisotope-bearing,ligand-containing small molecule (PIP-biotin-dextran prepared asdiscussed in part B hereof) was administered to this group of mice andalso to a group which had not received AO-Bt. The results of thisexperiment with respect to radiolabel uptake in tumor and clearance fromthe blood indicated that tumor-targeting of the radiolabeledbiotin-containing conjugate was retained while blood clearance wasenhanced, leading to an overall improvement in amount delivered totarget/amount located in serum. The AUC tumor/AUC blood with clearingagent was 6.87, while AUC tumor/AUC blood without clearing agent was4.45. Blood clearance of the circulating MAb-StrAv conjugate wasenhanced with the use of clearing agent. The clearing agent wasradiolabeled in a separate group of animals and found to bind directlyto tumor at very low levels (1.7 pmol/g at a dose of 488 total pmoles(0.35% ID/g), indicating that it does not significantly compromise theability of tumor-bound MAb-StrAv to bind subsequently administeredradiolabeled ligand.

[0653] B. Preparation Protocol for PIP-Biotin-Dextran. A solution of 3.0mg biotin-dextran, lysine fixable (BDLF, available from Sigma ChemicalCo., St. Louis, Mo., 70,000 dalton molecular weight with approximately18 biotins/molecule) in 0.3 ml PBS and 0.15 ml 1 M sodium carbonate, pH9.25, was added to a dried residue (1.87 mCi) of N-succinimidylp-I-125-iodobenzoate prepared in accordance with Wilbur, et al., J.Nucl. Med., 30: 216-226, 1989.

[0654] C. Dosing Optimization of AO-Bt. Tumored mice receiving StrAv-MAbas above, were injected with increasing doses of AO-Bt (0 micrograms, 20micrograms, 50 micrograms, 100 micrograms and 200 micrograms). Tumoruptake of I-131-PIP-biocytin (5.7 micrograms, administered 2 hours afterAO-Bt administration) was examined. Increasing doses of AO-Bt had noeffect on tumor localization of MAb-StrAv. Data obtained 44 hours afterAO-Bt administration showed the same lack of effect. This data indicatesthat AO-Bt dose not cross-link and internalize MAb-StrAv on the tumorsurface, as had been noted for avidin administered followingbiotinylated antibody.

[0655] PIP-biocytin tumor localization was inhibited at higher doses ofAO-Bt. This effect is most likely due to reprocessing and distributionto tumor of biotin used to derivatize AO-Bt. Optimal tumor to bloodratios (% injected dose of radiolabeled ligand/gram weight of tumordivided by % injected dose of radioligand/gram weight of blood wereachieved at the 50 microgram dose of AO-Bt. Biodistributions conductedfollowing completion of the protocols employing a 50 microgram AO-Btdose revealed low retention of radiolabel in all non-target tissues (1.2pmol/g in blood; 3.5 pmol/gram in tail; 1.0 pmol/g in lung; 2.2 pmol/gin liver; 1.0 pmol/g is spleen; 7.0 pmol/g in stomach; 2.7 pmol/g inkidney; and 7.7 pmol/g in intestine). With 99.3 pmol/g in tumor, theseresults indicate effective decoupling of the PIP-biocytinbiodistribution from that of the MAb-StrAv at all sites except tumor.This decoupling occurred at all clearing agent doses in excess of 50micrograms as well. Decreases in tumor localization of PIP-biocytin wasthe significant result of administering clearing agent doses in excessof 50 micrograms. In addition, the amount of PIP-biocytin in non-targettissues 44 hours after administration was identical to localizationresulting from administration of PIP-biocytin alone (except for tumor,where negligible accretion was seen when PIP-biocytin was administeredalone), indicating effective decoupling.

[0656] D. Further Investigation of Optimal Clearing Agent Dose. Tumoredmice injected with MAb-StrAv at time 0 as above; 50 micrograms of AO-Btat time 22 hours; and 545 microcuries of I-131-PIP-biocytin at time 25hours. Whole body radiation was measured and compared to that of animalsthat had not received clearing agent. 50 micrograms of AO-Bt wasefficient in allowing the injected radioactivity to clear from theanimals unimpeded by binding to circulating MAb-StrAv conjugate. Tumoruptake of I-131-PIP-biocytin was preserved at the 50 microgram clearingagent dose, with AUC tumor/AUC blood of 30:1 which is approximately15-fold better than the AUC tumor/AUC blood achieved in conventionalantibody-radioisotope therapy using this model.

[0657] E. Galactose- and Biotin-Derivatization of Human Serum Albumin(HSA). HSA was evaluated because it exhibits the advantages of beingboth inexpensive and non-immunogenic. HSA was derivatized with varyinglevels of biotin (1- about 9 biotins/molecule) via analogous chemistryto that previously described with respect to AO. More specifically, to asolution of HSA available from Sigma Chemical Co. (5-10 mg/ml in PBS)was added 10% v/v 0.5 M sodium borate buffer, pH 8.5, followed bydropwise addition of a DMSO solution of NHS-LC-biotin (Sigma ChemicalCo.) to the stirred solution at the desired molar offering (relativemolar equivalents of reactants). The final percent DMSO in the reactionmixture should not exceed 5%. After stirring for 1 hour at roomtemperature, the reaction was complete. A 90% incorporation efficiencyfor biotin on HSA was generally observed. As a result, if 3 molarequivalences of the NHS ester of LC-biotin was introduced, about 2.7biotins per HSA molecule were obtained. Unreacted biotin reagent wasremoved from the biotin-derivatized HSA using G-25 size exclusionchromatography. Alternatively, the crude material may be directlygalactosylated. The same chemistry is applicable for biotinylatingnon-previously biotinylated dextran.

[0658] HSA-biotin was then derivatized with from 12 to 15galactoses/molecule. Galactose derivatization of the biotinylated HSAwas performed according to the procedure of Lee, et al., Biochemistry,15: 3956, 1976. More specifically, a 0.1 M methanolic solution ofcyanomethyl-2,3,4,6-tetra-O-acetyl-l-thio-D-galactopyranoside wasprepared and reacted with a 10% v/v 0.1 M NaOMe in methanol for 12 hoursto generate the reactive galactosyl thioimidate. The galactosylation ofbiotinylated HSA began by initial evaporation of the anhydrous methanolfrom a 300 fold molar excess of reactive thioimidate. Biotinylated HSAin PBS, buffered with 10% v/v 0.5 M sodium borate, was added to the oilyresidue. After stirring at room temperature for 2 hours, the mixture wasstored at 4° C. for 12 hours. The galactosylated HSA-biotin was thenpurified by G-25 size exclusion chromatography or by buffer exchange toyield the desired product. The same chemistry is exploitable togalactosylating dextran. The incorporation efficiency of galactose onHSA is approximately 10%.

[0659] 70 micrograms of Galactose-HSA-Biotin (G-HSA-B), with 12-15galactose residues and 9 biotins, was administered to mice which hadbeen administered 200 micrograms of StrAv-MAb or 200 microliters of PBS24 hours earlier. Results indicated that G-HSA-B is effective inremoving StrAv-MAb from circulation. Also, the pharmacokinetics ofG-HSA-B is unperturbed and rapid in the presence or absence ofcirculating MAb-StrAv.

[0660] F. Non-Protein Clearing Agent. A commercially available form ofdextran, molecular weight of 70,000 daltons, pre-derivatized withapproximately 18 biotins/molecule and having an equivalent number offree primary amines was studied. The primary amine moieties werederivatized with a galactosylating reagent, substantially in accordancewith the procedure therefor described above in the discussion ofHSA-based clearing agents, at a level of about 9 galactoses/molecule.The molar equivalence offering ratio of galactose to HSA was about300:1, with about one-third of the galactose being converted to activeform. 40 Micrograms of galactose-dextran-biotin (GAL-DEX-BT) was theninjected i.v. into one group of mice which had received 200 microgramsMAb-StrAv conjugate intravenously 24 hours earlier, while 80 microgramsof GAL-DEX-BT was injected into other such mice. GAL-DEX-BT was rapidand efficient at clearing StrAv-MAb conjugate, removing over 66% ofcirculating conjugate in less than 4 hours after clearing agentadministration. An equivalent effect was seen at both clearing agentdoses, which correspond to 1.6 (40 micrograms) and 3.2 (80 micrograms)times the stoichiometric amount of circulating StrAv conjugate present.

[0661] G. Dose Ranging for G-HSA-B Clearing Agent. Dose ranging studiesfollowed the following basic format:

[0662] 200 micrograms MAb-StrAv conjugate administered;

[0663] 24 hours later, clearing agent administered; and

[0664] 2 hours later, 5.7 micrograms PIP-biocytin administered.

[0665] Dose ranging studies were performed with the G-HSA-B clearingagent, starting with a loading of 9 biotins per molecule and 12-15galactose residues per molecule. Doses of 20, 40, 70 and 120 microgramswere administered 24 hours after a 200 microgram dose of MAb-StrAvconjugate. The clearing agent administrations were followed 2 hourslater by administration of 5.7 micrograms of I-131-PIP-biocytin. Tumoruptake and blood retention of PIP-biocytin was examined 44 hours afteradministration thereof (46 hours after clearing agent administration).The results showed that a nadir in blood retention of PIP-biocytin wasachieved by all doses greater than or equal to 40 micrograms of G-HSA-B.A clear, dose-dependent decrease in tumor binding of PIP-biocytin ateach increasing dose of G-HSA-B was present, however. Since nodose-dependent effect on the localization of MAb-StrAv conjugate at thetumor was observed, this data was interpreted as being indicative ofrelatively higher blocking of tumor-associated MAb-StrAv conjugate bythe release of biotin from catabolized clearing agent. Similar resultsto those described earlier for the asialoorosomucoid clearing agentregarding plots of tumor/blood ratio were found with respect to G-HSA-B,in that an optimal balance between blood clearance and tumor retentionoccurred around the 40 microgram dose.

[0666] Because of the relatively large molar amounts of biotin thatcould be released by this clearing agent at higher doses, studies wereundertaken to evaluate the effect of lower levels of biotinylation onthe effectiveness of the clearing agent. G-HSA-B, derivatized witheither 9, 5 or 2 biotins/molecule, was able to clear MAb-StrAv conjugatefrom blood at equal protein doses of clearing agent. All levels ofbiotinylation yielded effective, rapid clearance of MAb-StrAv fromblood.

[0667] Comparison of these 9-, 5-, and 2-biotin-derivatized clearingagents with a single biotin G-HSA-B clearing agent was carried out intumored mice, employing a 60 microgram dose of each clearing agent. Thisexperiment showed each clearing agent to be substantially equallyeffective in blood clearance and tumor retention of MAb-StrAv conjugate2 hours after clearing agent administration. The G-HSA-B with a singlebiotin was examined for the ability to reduce binding of a subsequentlyadministered biotinylated small molecule (PIP-biocytin) in blood, whilepreserving tumor binding of PIP-biocytin to prelocalized MAb-StrAvconjugate. Measured at 44 hours following PIP-biocytin administration,tumor localization of both the MAb-StrAv conjugate and PIP-biocytin waswell preserved over a broad dose range of G-HSA-B with onebiotin/molecule (90 to 180 micrograms). A progressive decrease in bloodretention of PIP-biocytin was achieved by increasing doses of the singlebiotin G-HSA-B clearing agent, while tumor localization remainedessentially constant, indicating that this clearing agent, with a lowerlevel of biotinylation, is preferred. This preference arises because thesingle biotin G-HSA-B clearing agent is both effective at clearingMAb-StrAv over a broader range of doses (potentially eliminating theneed for patient-to-patient titration of optimal dose) and appears torelease less competing biotin into the systemic circulation than thesame agent having a higher biotin loading level.

[0668] Another way in which to decrease the effect of clearingagent-released biotin on active agent-biotin conjugate binding toprelocalized targeting moiety-streptavidin conjugate is to attach theprotein or polymer or other primary clearing agent component to biotinusing a retention linker. A retention linker has a chemical structurethat is resistant to agents that cleave peptide bonds and, optionally,becomes protonated when localized to a catabolizing space, such as alysosome. Preferred retention linkers of the present invention are shortstrings of D-amino acids or small molecules having both of thecharacteristics set forth above. An exemplary retention linker of thepresent invention is cyanuric chloride, which may be interposed betweenan epsilon amino group of a lysine of a proteinaceous primary clearingagent component and an amine moiety of a reduced and chemically alteredbiotin carboxy moiety (which has been discussed above) to form acompound of the structure set forth below.

[0669] When the compound shown above is catabolized in a catabolizingspace, the heterocyclic ring becomes protonated. The ring protonationprevents the catabolite from exiting the lysosome. In this manner,biotin catabolites containing the heterocyclic ring are restricted tothe site(s) of catabolism and, therefore, do not compete withactive-agent-biotin conjugate for prelocalized targetingmoiety-streptavidin target sites.

[0670] Comparisons of tumor/blood localization of radiolabeledPIP-biocytin observed in the G-HSA-B dose ranging studies showed thatoptimal tumor to background targeting was achieved over a broad doserange (90 to 180 micrograms), with the results providing the expectationthat even larger clearing agent doses would also be effective. Anotherkey result of the dose ranging experimentation is that G-HSA-B with anaverage of only 1 biotin per molecule is presumably only clearing theMAb-StrAv conjugate via the Ashwell receptor mechanism only, because toofew biotins are present to cause cross-linking and aggregation ofMAb-StrAv conjugates and clearing agents with such aggregates beingcleared by the reticuloendothelial system.

[0671] H. Tumor Targeting Evaluation Using G-HSA-B. The protocol forthis experiment was as follows:

[0672] Time 0: administer 400 micrograms MAb-StrAv conjugate;

[0673] Time 24 hours: administer 240 micrograms of G-HSA-B with onebiotin and 12-15 galactoses and

[0674] Time 26 hours: administer 6 micrograms of

[0675] Lu-177 is complexed with the DOTA chelate using known techniquestherefor.

[0676] Efficient delivery of the Lu-177-DOTA-biotin small molecule wasobserved, 20-25% injected dose/gram of tumor. These values areequivalent with the efficiency of the delivery of the MAb-StrAvconjugate. The AUC tumor/AUC blood obtained for this non-optimizedclearing agent dose was 300% greater than that achievable by comparabledirect MAb-radiolabel administration. In addition, the HSA-basedclearing agent is expected to exhibit a low degree of immunogenicity inhumans.

[0677] Kits containing one or more of the components described above arealso contemplated. For instance, radiohalogenated biotin may be providedin a sterile container for use in pretargeting procedures. Achelate-biotin conjugate provided in a sterile container is suitable forradiometallation by the consumer; such kits would be particularlyamenable for use in pretargeting protocols. Alternatively,radiohalogenated biotin and a chelate-biotin conjugate may be vialed ina non-sterile condition for use as a research reagent.

[0678] From the foregoing, it will be appreciated that, althoughspecific embodiments of the invention have been described herein forpurposes of illustration, various modifications may be made withoutdeviating from the spirit and scope of the invention. Accordingly, theinvention is not limited except as by the appended claims.

1-15. (Cancelled)
 16. A biotin-DOTA conjugate of the following formula:

wherein L is a linker of the formula

wherein R³ is hydrogen; an amine; lower alkyl; a hydroxy-, sulfate- orphosphonate-substituted lower alkyl; a glucuronide; or aglucuronide-derivatized amino acid; R⁴ is hydrogen, lower alkyl or

n ranges from 0-4, wherein R³ and R⁴ cannot both be hydrogen.
 17. Abiotin-DOTA conjugate according to claim 16 wherein R⁴ is lower alkyl.18. A biotin-DOTA conjugate according to claim 16 wherein R³ ishydrogen; R⁴ is CH₃; and n is
 4. 19. A biotin-DOTA conjugate accordingto claim 16 wherein R³ is hydrogen; R⁴ is CH₃; and n is 0.